Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions

ABSTRACT

Libraries of immunoglobulins which each have one or more amino acid modifications in at least one structural loop region of such immunoglobulins, where the modified loop region specifically binds to an epitope of an antigen to which an unmodified immunoglobulin does not significantly bind.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/149,871 filed on Feb. 2, 2012, which is a continuation of U.S. patentapplication Ser. No. 11/722,517, filed on Jun. 21, 2007, which is theU.S. national phase under 35 U.S.C. § 371 of PCT InternationalApplication No. PCT/EP2006/050059, filed on Jan. 5, 2006, which claimsthe benefit of priority under 35 U.S.C. 119(e) to U.S. ProvisionalPatent Application No. 60/641,144, filed on Jan. 5, 2005. The entirecontents of the foregoing patent applications are incorporated herein byreference in their entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The entire contents of a Sequence Listing titled 3906_1036_seq_listing,”created on Mar. 31, 2017 and having a size of 64 kilobytes, is beingfiled herewith in electronic form in connection with the presentapplication, and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to libraries of immunoglobulins which eachhave one or more amino acid modifications in at least one structuralloop region of such immunoglobulins, wherein the modified loop regionspecifically binds to an epitope of an antigen to which an unmodifiedimmunoglobulin does not significantly bind.

BACKGROUND OF THE INVENTION

The general field is the engineering of proteins with the aim to impartthem with specific binding properties. More specifically, the engineeredproteins of relevance here are immunoglobulins (antibodies), and evenmore specifically, single domains or pairs or combinations of singledomains of immunoglobulins. The specific binding properties ofimmunoglobulins are important features since they control theinteraction with other molecules such as antigens, and renderimmunoglobulins useful for diagnostic and therapeutic applications.

The basic antibody structure will be explained here using as example anintact IgG1 immunoglobulin.

Two identical heavy (H) and two identical light (L) chains combine toform the Y-shaped antibody molecule. The heavy chains each have fourdomains. The amino terminal variable domains (VH) are at the tips of theY. These are followed by three constant domains: CH1, CH2, and thecarboxy terminal CH3, at the base of the Y's stem. A short stretch, theswitch, connects the heavy chain variable and constant regions. Thehinge connects CH2 and CH3 (the Fc fragment) to the remainder of theantibody (the Fab fragments). One Fc and two identical Fab fragments canbe produced by proteolytic cleavage of the hinge in an intact antibodymolecule. The light chains are constructed of two domains, variable (VL)and constant (CL), separated by a switch.

Disulfide bonds in the hinge region connect the two heavy chains. Thelight chains are coupled to the heavy chains by additional disulfidebonds. Asn-linked carbohydrate moieties are attached at differentpositions in constant domains depending on the class of immunoglobulin.For IgG1 two disulfide bonds in the hinge region, between Cys235 andCys238 pairs, unite the two heavy chains. The light chains are coupledto the heavy chains by two additional disulfide bonds, between Cys229sin the CH1 domains and Cys214s in the CL domains. Carbohydrate moietiesare attached to Asn306 of each CH2, generating a pronounced bulge in thestem of the Y.

These features have profound functional consequences. The variableregions of both the heavy and light chains (VH) and (VL) lie at the“tips” of the Y, where they are positioned to react with antigen. Thistip of the molecule is the side on which the N-terminus of the aminoacid sequence is located. The stem of the Y projects in a way toefficiently mediate effector functions such as the activation ofcomplement and interaction with Fc receptors, or ADCC and ADCP. Its CH2and CH3 domains bulge to facilitate interaction with effector proteins.The C-terminus of the amino acid sequence is located on the oppositeside of the tip, which can be termed “bottom” of the Y. The structure ofan intact IgG1 is illustrated in FIG. 1 a.

Two types of light chain, termed lambda (λ) and kappa (κ), are found inantibodies. A given immunoglobulin either has κ chains or λ chains,never one of each. No functional difference has been found betweenantibodies having λ or κ light chains.

The structural organization of the main human immunoglobulin classmonomers is shown in FIG. 1b . The classes differ in the composition andsequence of their respective heavy chains. Both IgM and IgE lack a hingeregion but each contains an extra heavy-chain domain (CH4). Numbers andlocations of the disulfide bonds (lines) linking the chains differbetween the isotypes. They also differ in the distribution of N-linkedcarbohydrate groups, symbolically shown as circles.

Each domain in an antibody molecule has a similar structure of two betasheets packed tightly against each other in a compressed antiparallelbeta barrel. This conserved structure is termed the immunoglobulin fold.The immunoglobulin fold of constant domains contains a 3-stranded sheetpacked against a 4-stranded sheet. The fold is stabilized by hydrogenbonding between the beta strands of each sheet, by hydrophobic bondingbetween residues of opposite sheets in the interior, and by a disulfidebond between the sheets. The 3-stranded sheet comprises strands C, F,and G, and the 4-stranded sheet has strands A, B, E, and D. The lettersA through G denote the sequential positions of the beta strands alongthe amino acid sequence of the immunoglobulin fold.

The fold of variable domains has 9 beta strands arranged in two sheetsof 4 and 5 strands. The 5-stranded sheet is structurally homologous tothe 3-stranded sheet of constant domains, but contains the extra strandsC′ and C″. The remainder of the strands (A, B, C, D, E, F, G) have thesame topology and similar structure as their counterparts in constantdomain immunoglobulin folds. A disulfide bond links strands B and F inopposite sheets, as in constant domains. The immunoglobulin fold isillustrated in FIG. 2 for a constant and a variable domain of animmunoglobulin.

The variable domains of both light and heavy immunoglobulin chainscontain three hypervariable loops, or complementarity-determiningregions (CDRs). The three CDRs of a V domain (CDR1, CDR2, CDR3) clusterat one end of the beta barrel. The CDRs are loops that connect betastrands B-C, C′-C″, and F-G of the immunoglobulin fold. The residues inthe CDRs vary from one immunoglobulin molecule to the next, impartingantigen specificity to each antibody.

The VL and VH domains at the tips of antibody molecules are closelypacked such that the 6 CDRs (3 on each domain) cooperate in constructinga surface (or cavity) for antigen-specific binding. The natural antigenbinding site of an antibody thus is composed of the loops which connectstrands B-C, C′-C″, and F-G of the light chain variable domain andstrands B-C, C′-C″, and F-G of the heavy chain variable domain.

Using the 3D structure of a protein as an aid for design, amino acidresidues located on the surface of many proteins have been randomizedusing the core structure of the protein as scaffold. Examples for thisstrategy are described or reviewed in the following referencesincorporated herein by reference: Nygren P A, Uhlen M., Curr Opin StructBiol. (1997) 7:463-9; Binz H K, Amstutz P, Kohl A, Stumpp M T, Briand C,Forrer P, Grutter M G, Pluckthun A. Nat Biotechnol. (2004) 22:575-82;Vogt M, Skerra A. Chembiochem. (2004) 5:191-9; U.S. Pat. No. 6,562,617.

The basic principle of this technique is based on the observation thatmany proteins have a stable core, formed by specific arrangements ofsecondary structure elements such as beta sheets or alpha helices, whichare interconnected by structures such as loops, turns, or random coils.Typically, these latter three structure elements are less crucial forthe overall structure of the protein, and amino acid residues in thesestructure elements can be exchanged often without destroying the generalfold of the protein. A naturally occurring example for this designprinciple are the CDRs of antibodies. Artificial examples includelipocalins, ankyrins and other protein scaffolds.

The loops which are not CDR-loops in a native immunoglobulin do not haveantigen binding or epitope binding specificity but contribute to thecorrect folding of the entire immunoglobulin molecule and/or itseffector or other functions and are therefore called structural loopsfor the purpose of this invention.

In U.S. Pat. No. 6,294,654 it is shown that altered antibodies can bemade in which a peptide antigen can be incorporated into a non-CDR loopof an antibody (Ab) in the CH1 region between the hinge region and thevariable region, and the resulting Ab can be taken up in an APC so thatthe peptide antigen is presented on the surface of the APC in thecontext of MHC II, and thereby produce an immune response. Theseinserted peptides are epitopes and the overall structure of the carriermolecule is not important. It was demonstrated that a ras peptide can beplaced on a (non-CDR) loop of an Immunoglobulin and the Immunoglobulinstill be secreted. There is stringent “quality control” in the cellswhich prevent the Immunoglobulin from being secreted unless it isproperly folded, and altering the amino acid sequence of the loop mightcause the protein to fold into a structure which the cell would detectas incorrect, and hence degrade it. Thus, besides the examples shown itwas considered to be difficult to further modify the structural loopswithout changing the nature of the Immunoglobulin.

US Pat Appl 2004/0101905 describes binding molecules comprising a targetbinding site and a Fc effector peptide. The Fc effector peptide is apeptide which interacts with effector molecule. The insertion of aneffector peptide into a non-CDR loop of a CH1-domain of animmunoglobulin fragment has been shown.

Fc effector peptides are structures which are naturally occurring innon-CDR loops of antibodies and are therefore expected not to disturbthe structure of the immunoglobulin if grafted to onto differentequivalent locations in an immunoglobulin.

Nevertheless every peptide grafted into a non-CDR loop according to thisdisclosure has a high chance of being inactive by the differentstructural environment it has been selected.

It is stated in both prior art documents mentioned above that it isdifficult to insert peptides into the loop that should retain itsstructure and function, as it is critical not to disturb theimmunoglobulin folding structure as this is important for function andsecretion.

US Patent Applications 2004/0132101 and 2005/0244403 describe mutantimmunoglobulins with altered binding affinity to an effector ligand,which are natural ligands for structural loops of antibodies. In thisdocument a number of mutations in various regions across the entireimmunoglobulin molecule are described which influence the effectorfunction of the entire antibody.

Other prior art documents show that the immunoglobulin like scaffold hasbeen employed so far for the purpose of manipulating the existingantigen binding site, thereby introducing novel binding properties. Sofar however, only the CDR regions have been engineered for antigenbinding, in other words, in the case of the immunoglobulin fold, onlythe natural antigen binding site has been modified in order to changeits binding affinity or specificity. A vast body of literature existswhich describes different formats of such manipulated immunoglobulins,frequently expressed in the form of single-chain Fv fragments (scFv) orFab fragments, either displayed on the surface of phage particles orsolubly expressed in various prokaryotic or eukaryotic expressionsystems. Among the leading authors in the field are Greg Winter, AndreasPlückthun and Hennie Hoogenboom.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide immunoglobulins withnew antigen binding sites introduced, and methods for engineering andmanufacturing said immunoglobulins.

Therefore, the present invention relates to a method for engineering animmunoglobulin comprising at least one modification in a structural loopregion of said immunoglobulin and determining the binding of saidimmunoglobulin to an epitope of an antigen, wherein the unmodifiedimmunoglobulin does not significantly bind to said epitope, comprisingthe steps of:

-   -   providing a nucleic acid encoding an immunoglobulin comprising        at least one structural loop region,    -   modifying at least one nucleotide residue of at least one of        said structural loop regions,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with an        epitope, and    -   determining whether said modified immunoglobulin binds to said        epitope.

In particular, the present invention relates to a method for engineeringan immunoglobulin binding specifically to an epitope of an antigenselected from the group consisting of allergens, tumor associatedantigens, self antigens, enzymes, bacterial antigens, fungal antigens,protozooal antigen and viral antigens. Through the modification in thestructural loop region the immunoglobulin may be engineered bind to theepitope. In a preferred embodiment the immunoglobulin is bindingspecifically to at least two such epitopes, that differ from each other,either of the same antigen or of different antigens.

For example, the method according to the invention refers to engineeringan immunoglobulin binding specifically to at least one first epitope andcomprising at least one modification in at least one structural loopregion of said immunoglobulin and determining the specific binding ofsaid at least one loop region to at least one second epitope, theepitope being selected from the group of antigens as mentioned above,wherein the unmodified structural loop region (non-CDR region) does notspecifically bind to said at least one second epitope, comprising thesteps of:

-   -   providing a nucleic acid encoding an immunoglobulin binding        specifically to at least one first epitope comprising at least        one structural loop region,    -   modifying at least one nucleotide residue of at least one of        said loop regions encoded by said nucleic acid,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with said at        least one second epitope, and    -   determining whether said modified immunoglobulin binds        specifically to the second epitope.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated in the following figuresand examples without being restricted thereto.

FIG. 1a shows the structure of an intact IgG1. Domains are indicatedwith arrows.

FIG. 1b illustrates the structural organization of the main humanimmunoglobulin isotype monomers. Disulfide bonds are shown as lines,N-linked carbohydrate groups are shown as circles.

FIG. 2 shows the immunoglobulin fold for a constant (left) and avariable (right) domain of an immunoglobulin. Beta strands are indicatedby arrows

FIG. 3 shows a molecular model of the engineered CH3 domain according tothe present invention, with the randomized part indicated by a solventaccessible surface. The surface is circled.

FIG. 4 shows a schematic presentation of the PCRs used for production ofthe fragments used for assembly of the mutated CH3 domain. PCR primersare indicated by arrows with their respective 5′-3′ orientation, andvertical lines indicate the approximate positions of the introducedrestriction sites which were used for assembly of the mutated gene. Thefollowing restriction sites are contained on the primers for ligationsof the PCR fragments: CH3LNCO: NcoI; CH3LSAC and CH3CSAC: SacI; CH3CHINand CH3RHIN: HindIII; CH3RNOT: NotI.

FIG. 5 shows some examples of how the immunoglobulin domains of thecurrent application could be used. Randomized regions are indicated by astar symbol. Specificities of the randomized regions in one molecule caneither be identical or different.

FIG. 6 shows a schematic presentation of the design of the bispecificengineered CH3 domain. Names of primers are given in boxes and arrowsindicate the direction in which the primers are elongated. Boxes withsloping lines indicate the relative positions of regions that arerandomized in this construct, boxes with vertical lines indicate therelative positions of regions that were introduced for generation ofclone C24, and restriction sites used for the cloning procedure aregiven.

FIG. 7 shows a schematic presentation of the design of the bispecificengineered CH3 domain. The double stranded nucleotide sequence and itstranslation is shown of the basic design of the bispecific engineeredCH3 domain directed to recombinant human Erythropoietin (rhEPO) and henegg lysozyme. The gaps in the amino acid sequence encoded by the doublestranded nucleotide sequence indicates randomized regions in order togenerate the bispecific construct, including regions in which thesequence was randomized in order to generate the hen egg lysozymespecific clone C24. The amino acid segments intervening said gaps fromleft to right starting in the top row are: REPQVYTLPPSRDEL (residues 4through 18 of SEQ ID NO:13); QVSLTCLVKGF (residues 22 through 32 of SEQID NO:13); IAVEWESNGQPENNYKTTPPVLDSDG (residues 37 through 62 of SEQ IDNO:13); SFFLYSKLTV (residues 63 through 72 of SEQ ID NO:13); RW residues81 through 82 of SEQ ID NO:13; GNVFSCSVM (residues 85 through 93 of SEQID NO:13); and LHNHYTQKSLSLSPGKAAA (residues 97 through 115 of SEQ IDNO:13).

FIG. 8 shows the sequence listing of the sequences disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention preferably refers to at least onemodification in at least one structural loop region of saidimmunoglobulin and determining the specific binding of said at least oneloop region to at least one antigen selected from the group consistingof allergens, tumor associated antigens, self antigens, enzymes,bacterial antigens, fungal antigens, viral antigens and protozooalantigens, wherein the immunoglobulin containing an unmodified structuralloop region does not specifically bind to said at least one antigen.

The term “immunoglobulins” according to the present invention to bemodified (as used herein the terms immunoglobulin and antibody areinterchangeable) may exhibit mono- or multi-specific, or multivalentbinding properties, at least two, preferably at least three specificbinding sites for epitopes of e.g. antigens, effectormolecules/proteins. Immunoglobulins according to the invention are alsofunctional fragments accepted in the art, such as Fc, Fab, scFv, singlechain dimers of CH/CL domains, Fv, or other derivatives or combinationsof the immunoglobulins, domains of the heavy and light chains of thevariable region (such as Fd, Vl, Vk, Vh) and the constant region of anintact antibody such as CH1, CH2, CH3, CH4, Cl and Ck, as well asmini-domains consisting of two beta-strands of an immunoglobulin domainconnected by a structural loop.

It is understood that the term “immunoglobulin”, “modifiedimmunoglobulin” or “immunoglobulin according to the invention” includesa derivative of immunoglobulins as well. A derivative is any combinationof one or more immunoglobulins of the invention and or a fusion proteinin which any domain or minidomain of the immunoglobulin of the inventionmay be fused at any position of one or more other proteins (such asother immunoglobulins, ligands, scaffold proteins, enzymes toxins andthe like). A derivative of the immunoglobulin of the invention may alsobe obtained by binding to other substances by various chemicaltechniques such as covalent coupling, electrostatic interaction,disulphide bonding etc.

The other substances bound to the immunoglobulins may be lipids,carbohydrates, nucleic acids, organic and anorganic molecules or anycombination thereof (e.g. PEG, prodrugs or drugs). A derivative is alsoan immunoglobulin with the same amino acid sequence but made completelyor partly from non-natural or chemically modified amino acids.

The engineered molecules according to the present invention will beuseful as stand-alone proteins as well as fusion proteins orderivatives, most typically fused in such a way as to be part of largerantibody structures or complete antibody molecules, or parts thereofsuch as Fab fragments, Fc fragments, Fv fragments and others. It will bepossible to use the engineered proteins to produce molecules which aremonospecific, bispecific, trispecific, and maybe even carry morespecificities at the same time, and it will be possible at the same timeto control and preselect the valency of binding at the same timeaccording to the requirements of the planned use of such molecules.

According to the present invention, binding regions to antigens orantigen binding sites of all kinds of allergens, tumor associatedantigens, self antigens, enzymes, bacterial antigens, fungal antigens,protozooal antigen and viral antigens, may be introduced into astructural loop of a given antibody structure.

The term “antigen” according to the present invention shall meanmolecules or structures known to interact or capable of interacting withthe CDR-loop region of immunoglobulins. Structural loop regions of theprior art do not interact with antigens but rather contribute to theoverall structure and/or to the binding to effector molecules.

The term “allergens, tumor associated antigens, self antigens, enzymes,bacterial antigens, fungal antigens, protozooal antigen and viralantigens” according to the present invention shall include all allergensand antigens capable of being recognised by an antibody structure, andfragments of such molecules (especially substructures generally referredto as “epitopes” (e.g. B-cell epitopes)), as long as they areimmunologically relevant, i.e. are also recognisable by natural ormonoclonal antibodies.

The term “epitope” according to the present invention shall mean amolecular structure which may completely make up a specific bindingpartner or be part of a specific binding partner to the binding domainor the immunoglobulin of the present invention.

Chemically, an epitope may either be composes of a carbohydrate, apeptide, a fatty acid, a anorganic substance or derivatives thereof andany combinations thereof. If an epitope is a polypeptide, it willusually include at least 3 amino acids, preferably 8 to 50 amino acids,and more preferably between about 10-20 amino acids in the peptide.There is no critical upper limit to the length of the peptide, whichcould comprise nearly the full length of the polypeptide sequence.Epitopes can be either linear or conformational epitopes. A linearepitope is comprised of a single segment of a primary sequence of apolypeptide chain. Linear epitopes can be contiguous or overlapping.Conformational epitopes are comprised of amino acids brought together byfolding of the polypeptide to form a tertiary structure and the aminoacids are not necessarily adjacent to one another in the linearsequence.

Specifically, epitopes are at least part of diagnostically relevantmolecules, i.e. the absence or presence of an epitope in a sample isqualitatively or quantitatively correlated to either a disease or to thehealth status or to a process status in manufacturing or toenvironmental and food status. Epitopes may also be at least part oftherapeutically relevant molecules, i.e. molecules which can be targetedby the specific binding domain which changes the course of the disease.

Preferred “allergens, tumor associated antigens, self antigens, enzymes,bacterial antigens, fungal antigens, protozooal antigen and viralantigens,” are those allergens or antigens, which have already beenproven to be or are capable of being immunologically or therapeuticallyrelevant, especially those, for which a clinical efficacy has beentested.

On the other hand, according to another aspect of the present inventionalso other binding capacities may be introduced in the structural loopregions, e.g. binding capacities for small molecules, such as drugs orenzymes, catalytic sites of enzymes or enzyme substrates or for atransition state analog of an enzyme substrate.

Preferably the new antigen binding site in the structural loops isforeign to the unmodified immunoglobulin. Thus targets like effectormolecules or Fc-receptors are preferably excluded from the bindingmolecules and the specificity of the immunoglobulins according to theinvention.

Preferably, the new antigen binding sites in the structural loops areintroduced by substitution, deletion and/or insertion of theimmunoglobulin encoded by the selected nucleic acid.

According to another preferred embodiment of the present invention themodification of at least one nucleotide results in a substitution,deletion and/or insertion of the immunoglobulin encoded by said nucleicacid.

The modification of the at least one loop region may result in asubstitution, deletion and/or insertion of 1 or more amino acids,preferably a point mutation, exchange of whole loops, more preferred thechange of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 30 amino acids.

Also preferred is the site directed random mutation. With this methodone or more specific amino acid residues of the loop are exchanged orintroduced using randomly generated inserts into such structural loops.Alternatively preferred is the use of combinatorial approaches.

The at least one loop region is preferably mutated or modified byrandom, semi-random or, in particular, by site-directed randommutagenesis methods. These methods may be used to make amino acidmodifications at desired positions of the immunoglobulin of the presentinvention. In these cases positions are chosen randomly, or amino acidchanges are made using simplistic rules. For example all residues may bemutated to alanine, referred to as alanine scanning. Such methods may becoupled with more sophisticated engineering approaches that employselection methods to screen higher levels of sequence diversity. Apreferred method according to the invention refers to the randomlymodified nucleic acid molecule which comprises at least one nucleotiderepeating unit having the sequence 5′-NNS-3′, 5′-NNN-3′ or 5′-NNK-3′.

The randomly modified nucleic acid molecule may comprise the aboveidentified repeating units, which code for all known naturally occurringamino acids.

As is well known in the art, there are a variety of selectiontechnologies that may be used for the identification and isolation ofproteins with certain binding characteristics and affinities, including,for example, display technologies such as phage display, ribosomedisplay, cell surface display, and the like, as described below. Methodsfor production and screening of antibody variants are well known in theart. General methods for antibody molecular biology, expression,purification, and screening are described in Antibody Engineering,edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001; andHayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &Georgiou, 2000, Annu Rev Biomed Eng 2:339-76.

A “structural loop” or “non-CDR-loop” according to the present inventionis to be understood in the following manner: immunoglobulins are made ofdomains with a so called immunoglobulin fold. In essence, anti-parallelbeta sheets are connected by loops to form a compressed antiparallelbeta barrel. In the variable region, some of the loops of the domainscontribute essentially to the specificity of the antibody, i.e. thebinding to an antigen. These loops are called CDR-loops. All other loopsof antibody domains are rather contributing to the structure of themolecule and/or the effector function. These loops are defined herein asstructural loops or non-CDR-loops.

The nucleic acid molecules encoding the modified immunoglobulins (andalways included throughout the whole specification below: immunoglobulinfragments) may be cloned into host cells, expressed and assayed fortheir binding specificities. These practices are carried out usingwell-known procedures, and a variety of methods that may find use in thepresent invention are described in Molecular Cloning—A LaboratoryManual, 3.sup.rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, NewYork, 2001), and Current Protocols in Molecular Biology (John Wiley &Sons). The nucleic acids that encode the modified immunoglobulins of thepresent invention may be incorporated into an expression vector in orderto express said immunoglobulins. Expression vectors typically comprisean immunoglobulin operably linked, that is placed in a functionalrelationship, with control or regulatory sequences, selectable markers,any fusion partners, and/or additional elements. The modifiedimmunoglobulins of the present invention may be produced by culturing ahost cell transformed with nucleic acid, preferably an expressionvector, containing nucleic acid encoding the modified immunoglobulins,under the appropriate conditions to induce or cause expression of themodified immunoglobulins. The methods of introducing exogenous nucleicacid molecules into a host are well known in the art, and will vary withthe host used. Of course, also acellular or cell free expression systemsfor the expression of modified immunoglobulins may be employed.

In a preferred embodiment of the present invention, the modifiedimmunoglobulins are purified or isolated after expression. Modifiedimmunoglobulins may be isolated or purified in a variety of ways knownto those skilled in the art. Standard purification methods includechromatographic techniques, electrophoretic, immunological,precipitation, dialysis, filtration, concentration, and chromatofocusingtechniques. Purification can often be enabled by a particular fusionpartner. For example, antibodies may be purified using glutathione resinif a GST fusion is employed, Ni⁺² affinity chromatography if a His-tagis employed or immobilized anti-flag antibody if a flag-tag is used. Forgeneral guidance in suitable purification techniques, see AntibodyPurification: Principles and Practice, 3.sup.rd Ed., Scopes,Springer-Verlag, NY, 1994. Of course, it is also possible to express themodified immunoglobulins according to the present invention on thesurface of a host, in particular on the surface of a bacterial, insector yeast cell or on the surface of phages or viruses.

Modified immunoglobulins may be screened using a variety of methods,including but not limited to those that use in vitro assays, in vivo andcell-based assays, and selection technologies. Automation andhigh-throughput screening technologies may be utilized in the screeningprocedures. Screening may employ the use of a fusion partner or label,for example an enzyme, an immune label, isotopic label, or smallmolecule label such as a fluorescent or colorimetric dye or aluminogenic molecule.

In a preferred embodiment, the functional and/or biophysical propertiesof the immunoglobulins are screened in an in vitro assay. In a preferredembodiment, the antibody is screened for functionality, for example itsability to catalyze a reaction or its binding affinity to its target.

Assays may employ a variety of detection methods including but notlimited to chromogenic, fluorescent, luminescent, or isotopic labels.

As is known in the art, a subset of screening methods are those thatselect for favorable members of a library. The methods are hereinreferred to as “selection methods”, and these methods find use in thepresent invention for screening modified immunoglobulins. Whenimmunoglobulins libraries are screened using a selection method, onlythose members of a library that are favorable, that is which meet someselection criteria, are propagated, isolated, and/or observed. As willbe appreciated, because only the most fit variants are observed, suchmethods enable the screening of libraries that are larger than thosescreenable by methods that assay the fitness of library membersindividually. Selection is enabled by any method, technique, or fusionpartner that links, covalently or noncovalently, the phenotype ofimmunoglobulins with its genotype, that is the function of a antibodywith the nucleic acid that encodes it. For example the use of phagedisplay as a selection method is enabled by the fusion of librarymembers to the gene III protein. In this way, selection or isolation ofmodified immunoglobulins that meet some criteria, for example bindingaffinity to the immunoglobulin's target, also selects for or isolatesthe nucleic acid that encodes it. Once isolated, the gene or genesencoding modified immunoglobulins may then be amplified. This process ofisolation and amplification, referred to as panning, may be repeated,allowing favorable antibody variants in the library to be enriched.Nucleic acid sequencing of the attached nucleic acid ultimately allowsfor gene identification.

A variety of selection methods are known in the art that may find use inthe present invention for screening immunoglobulin libraries. Theseinclude but are not limited to phage display (Phage display of peptidesand antibodies: a laboratory manual, Kay et al., 1996, Academic Press,San Diego, Calif., 1996; Lowman et al., 1991, Biochemistry30:10832-10838; Smith, 1985, Science 228:1315-1317) and its derivativessuch as selective phage infection (Malmborg et al., 1997, J Mol Biol273:544-551), selectively infective phage (Krebber et al., 1997, J MolBiol 268:619-630), and delayed infectivity panning (Benhar et al., 2000,J Mol Biol 301:893-904), cell surface display (Witrrup, 2001, Curr OpinBiotechnol, 12:395-399) such as display on bacteria (Georgiou et al.,1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993, Trends Biotechnol11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648; June et al., 1998,Nat Biotechnol 16:576-80), yeast (Boder & Wittrup, 2000, Methods Enzymol328:430-44; Boder & Wittrup, 1997, Nat Biotechnol 15:553-557), andmammalian cells (Whitehorn et al., 1995, Bio/technology 13:1215-1219),as well as in vitro display technologies (Amstutz et al., 2001, CurrOpin Biotechnol 12:400-405) such as polysome display (Mattheakis et al.,1994, Proc Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes etal., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display (Roberts &Szostak, 1997, Proc Natl Acad Sci USA 94:12297-12302; Nemoto et al.,1997, FEBS Lett 414:405-408), and ribosome-inactivation display system(Zhou et al., 2002, J Am Chem Soc 124, 538-543).

Other selection methods that may find use in the present inventioninclude methods that do not rely on display, such as in vivo methodsincluding but not limited to periplasmic expression and cytometricscreening (Chen et al., 2001, Nat Biotechnol 19:537-542), the antibodyfragment complementation assay (Johnsson & Varshaysky, 1994, Proc NatlAcad Sci USA 91:10340-10344; Pelletier et al., 1998, Proc Natl Acad SciUSA 95:12141-12146), and the yeast two hybrid screen (Fields & Song,1989, Nature 340:245-246) used in selection mode (Visintin et al., 1999,Proc Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,selection is enabled by a fusion partner that binds to a specificsequence on the expression vector, thus linking covalently ornoncovalently the fusion partner and associated Fc variant librarymember with the nucleic acid that encodes them. For example, PCT WO00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO02/08023; PCT WO 01/28702; and PCT WO 02/07466 describe such a fusionpartner and technique that may find use in the present invention. In analternative embodiment, in vivo selection can occur if expression of theantibody imparts some growth, reproduction, or survival advantage to thecell.

A subset of selection methods referred to as “directed evolution”methods are those that include the mating or breeding of favourablesequences during selection, sometimes with the incorporation of newmutations. As will be appreciated by those skilled in the art, directedevolution methods can facilitate identification of the most favourablesequences in a library, and can increase the diversity of sequences thatare screened. A variety of directed evolution methods are known in theart that may find use in the present invention for screening antibodyvariants, including but not limited to DNA shuffling (PCT WO 00/42561A3; PCT WO 01/70947 A3), exon shuffling (U.S. Pat. No. 6,365,377;Kolkman & Stemmer, 2001, Nat Biotechnol 19:423-428), family shuffling(Crameri et al., 1998, Nature 391:288-291; U.S. Pat. No. 6,376,246),RACHITT™ (Coco et al., 2001, Nat Biotechnol 19:354-359; PCT WO02/06469), STEP and random priming of in vitro recombination (Zhao etal., 1998, Nat Biotechnol 16:258-261; Shao et al., 1998, Nucleic AcidsRes 26:681-683), exonuclease mediated gene assembly (U.S. Pat. Nos.6,352,842; 6,361,974), Gene Site Saturation Mutagenesis™ (U.S. Pat. No.6,358,709), Gene Reassembly™ (U.S. Pat. No. 6,358,709), SCRATCHY (Lutzet al., 2001, Proc Natl Acad Sci USA 98:11248-11253), DNA fragmentationmethods (Kikuchi et al., Gene 236:159-167), single-stranded DNAshuffling (Kikuchi et al., 2000, Gene 243:133-137), and AMEsystem™directed evolution antibody engineering technology (Applied MolecularEvolution) (U.S. Pat. Nos. 5,824,514; 5,817,483; 5,814,476; 5,763,192;5,723,323).

In a preferred embodiment, antibody variants are screened using one ormore cell-based or in vivo assays. For such assays, purified orunpurified modified immunoglobulins are typically added exogenously suchthat cells are exposed to individual immunoglobulins or pools ofimmunoglobulins belonging to a library. These assays are typically, butnot always, based on the function of the immunoglobulin; that is, theability of the antibody to bind to its target and mediate somebiochemical event, for example effector function, ligand/receptorbinding inhibition, apoptosis, and the like. Such assays often involvemonitoring the response of cells to the antibody, for example cellsurvival, cell death, change in cellular morphology, or transcriptionalactivation such as cellular expression of a natural gene or reportergene. For example, such assays may measure the ability of antibodyvariants to elicit ADCC, ADCP, or CDC. For some assays additional cellsor components, that is in addition to the target cells, may need to beadded, for example serum complement, or effector cells such asperipheral blood monocytes (PBMCs), NK cells, macrophages, and the like.Such additional cells may be from any organism, preferably humans, mice,rat, rabbit, and monkey. Immunoglobulins may cause apoptosis of certaincell lines expressing the target, or they may mediate attack on targetcells by immune cells which have been added to the assay. Methods formonitoring cell death or viability are known in the art, and include theuse of dyes, immunochemical, cytochemical, and radioactive reagents. Forexample, caspase staining assays may enable apoptosis to be measured,and uptake or release of radioactive substrates or fluorescent dyes suchas alamar blue may enable cell growth or activation to be monitored. Ina preferred embodiment, the DELFIA® EuTDA-based cytotoxicity assay(Perkin Elmer, MA) may be used. Alternatively, dead or damaged targetcells may be monitored by measuring the release of one or more naturalintracellular components, for example lactate dehydrogenase.Transcriptional activation may also serve as a method for assayingfunction in cell-based assays. In this case, response may be monitoredby assaying for natural genes or immunoglobulins which may beupregulated, for example the release of certain interleukins may bemeasured, or alternatively readout may be via a reporter construct.Cell-based assays may also involve the measure of morphological changesof cells as a response to the presence of modified immunoglobulins. Celltypes for such assays may be prokaryotic or eukaryotic, and a variety ofcell lines that are known in the art may be employed. Alternatively,cell-based screens are performed using cells that have been transformedor transfected with nucleic acids encoding the variants. That is,antibody variants are not added exogenously to the cells. For example,in one embodiment, the cell-based screen utilizes cell surface display.A fusion partner can be employed that enables display of modifiedimmunoglobulins on the surface of cells (Witrrup, 2001, Curr OpinBiotechnol, 12:395-399).

In a preferred embodiment, the immunogenicity of the modifiedimmunoglobulins may be determined experimentally using one or morecell-based assays. In a preferred embodiment, ex vivo T-cell activationassays are used to experimentally quantitate immunogenicity. In thismethod, antigen presenting cells and naive T cells from matched donorsare challenged with a peptide or whole antibody of interest one or moretimes. Then, T cell activation can be detected using a number ofmethods, for example by monitoring production of cytokines or measuringuptake of tritiated thymidine. In the most preferred embodiment,interferon gamma production is monitored using Elispot assays (Schmittelet. al., 2000, J. Immunol. Meth., 24: 17-24).

The biological properties of the modified immunoglobulins of the presentinvention may be characterized in cell, tissue, and whole organismexperiments. As is known in the art, drugs are often tested in animals,including but not limited to mice, rats, rabbits, dogs, cats, pigs, andmonkeys, in order to measure a drug's efficacy for treatment against adisease or disease model, or to measure a drug's pharmacokinetics,toxicity, and other properties. The animals may be referred to asdisease models. Therapeutics are often tested in mice, including but notlimited to nude mice, SCID mice, xenograft mice, and transgenic mice(including knockins and knockouts). Such experimentation may providemeaningful data for determination of the potential of the antibody to beused as a therapeutic. Any organism, preferably mammals, may be used fortesting. For example because of their genetic similarity to humans,monkeys can be suitable therapeutic models, and thus may be used to testthe efficacy, toxicity, pharmacokinetics, or other property of themodified immunoglobulins of the present invention. Tests of the inhumans are ultimately required for approval as drugs, and thus of coursethese experiments are contemplated. Thus the modified immunoglobulins ofthe present invention may be tested in humans to determine theirtherapeutic efficacy, toxicity, immunogenicity, pharmacokinetics, and/orother clinical properties.

The modified immunoglobulins of the present invention may find use in awide range of antibody products. In one embodiment the antibody variantof the present invention is used for therapy or prophylaxis, forpreparative or analytic use, as a diagnostic, an industrial compound ora research reagent, preferably a therapeutic. The antibody variant mayfind use in an antibody composition that is monoclonal or polyclonal. Ina preferred embodiment, the modified immunoglobulins of the presentinvention are used to kill target cells that bear the target antigen,for example cancer cells. In an alternate embodiment, the modifiedimmunoglobulins of the present invention are used to block, antagonize,or agonize the target antigen, for example by antagonizing a cytokine orcytokine receptor. In an alternately preferred embodiment, the modifiedimmunoglobulins of the present invention are used to block, antagonize,or agonize the target antigen and kill the target cells that bear thetarget antigen.

In an alternately preferred embodiment, the modified immunoglobulins ofthe present invention are used to block, antagonize, or agonize growthfactors or growth factor receptors and kill the target cells that bearor need the target antigen. In an alternately preferred embodiment, themodified immunoglobulins of the present invention are used to block,antagonize, or agonize enzymes and substrate of enzymes.

The modified immunoglobulins of the present invention may be used forvarious therapeutic purposes. In a preferred embodiment, an antibodycomprising the modified immunoglobulins is administered to a patient totreat a specific disorder. A “patient” for the purposes of the presentinvention includes both humans and other animals, preferably mammals andmost preferably humans. By “specific disorder” herein is meant adisorder that may be ameliorated by the administration of apharmaceutical composition comprising a modified immunoglobulin of thepresent invention.

In one embodiment, a modified immunoglobulin according to the presentinvention is the only therapeutically active agent administered to apatient. Alternatively, the modified immunoglobulin according thepresent invention are administered in combination with one or more othertherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, or other therapeutic agents. The modifiedimmunoglobulins may be administered concomitantly with one or more othertherapeutic regimens. For example, an antibody variant of the presentinvention may be administered to the patient along with chemotherapy,radiation therapy, or both chemotherapy and radiation therapy. In oneembodiment, the modified immunoglobulins of the present invention may beadministered in conjunction with one or more antibodies, which may ormay not comprise a antibody variant of the present invention. Inaccordance with another embodiment of the invention, the modifiedimmunoglobulins of the present invention and one or more otheranti-cancer therapies are employed to treat cancer cells ex vivo. It iscontemplated that such ex vivo treatment may be useful in bone marrowtransplantation and particularly, autologous bone marrowtransplantation. It is of course contemplated that the antibodies of theinvention can be employed in combination with still other therapeutictechniques such as surgery.

A variety of other therapeutic agents may find use for administrationwith the modified immunoglobulins of the present invention. In oneembodiment, the modified immunoglobulin is administered with ananti-angiogenic agent, which is a compound that blocks, or interferes tosome degree, the development of blood vessels. The anti-angiogenicfactor may, for instance, be a small molecule or a protein, for examplean antibody, Fc fusion, or cytokine, that binds to a growth factor orgrowth factor receptor involved in promoting angiogenesis. The preferredanti-angiogenic factor herein is an antibody that binds to VascularEndothelial Growth Factor (VEGF). In an alternate embodiment, themodified immunoglobulin is administered with a therapeutic agent thatinduces or enhances adaptive immune response, for example an antibodythat targets CTLA-4. In an alternate embodiment, the modifiedimmunoglobulin is administered with a tyrosine kinase inhibitor, whichis a molecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. In an alternate embodiment, the modifiedimmunoglobulins of the present invention are administered with acytokine. By “cytokine” as used herein is meant a generic term forproteins released by one cell population that act on another cell asintercellular mediators including chemokines.

Pharmaceutical compositions are contemplated wherein modifiedimmunoglobulins of the present invention and one or more therapeuticallyactive agents are formulated. Formulations of the antibody variants ofthe present invention are prepared for storage by mixing saidimmunoglobulin having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980),in the form of lyophilized formulations or aqueous solutions. Theformulations to be used for in vivo administration are preferablysterile. This is readily accomplished by filtration through sterilefiltration membranes or other methods. The modified immunoglobulins andother therapeutically active agents disclosed herein may also beformulated as immunoliposomes, and/or entrapped in microcapsules

Administration of the pharmaceutical composition comprising a modifiedimmunoglobulin of the present invention, preferably in the form of asterile aqueous solution, may be done in a variety of ways, including,but not limited to, orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, topically (e.g., gels, salves, lotions,creams, etc.), intraperitoneally, intramuscularly, intrapulmonary (e.g.,AERx™ inhalable technology commercially available from Aradigm, orInhance™ pulmonary delivery system commercially available from InhaleTherapeutics), vaginally, parenterally, rectally, or intraocularly.

As used herein, the term “specifically binds” refers to a bindingreaction which is determinative of the cognate ligand of interest in aheterogeneous population of molecules. Thus, under designated conditions(e.g. immunoassay conditions in the case of an immunoglobulin), thespecified antibody binds to its particular “target” and does not bind ina significant amount to other molecules present in a sample. Comparableto CDRs of antibodies the modified structural loop regions are antigen-or molecule-binding protein moieties and not antigens as such.

The term “expression system” refers to nucleic acid molecules containinga desired coding sequence and control sequences in operable linkage, sothat hosts transformed or transfected with these sequences are capableof producing the encoded proteins. In order to effect transformation,the expression system may be included on a vector; however, the relevantDNA may than also be integrated into the host chromosome.

According to a preferred embodiment of the present invention theexpression system comprises a vector. Any expression vector known in theart may be used for this purpose as appropriate.

The modified immunoglobulin is preferably expressed in a host,preferably in a bacterial, a yeast, a plant cell, in an animal cell orin a plant or animal.

A wide variety of appropriate host cells may be used to express themodified immunoglobulin, including but not limited to mammalian cells(animal cells), plant cells, bacteria (e.g. Bacillus subtilis,Escherichia coli), insect cells, and yeast (e.g. Pichia pastoris,Saccharomyces cerevisiae). For example, a variety of cell lines that mayfind use in the present invention are described in the ATCC cell linecatalog, available from the American Type Culture Collection.Furthermore, also plants and animals may be used as hosts for theexpression of the immunoglobulin according to the present invention. Theexpression as well as the transfection vectors or cassettes may beselected according to the host used.

Of course also acellular or cell free protein expression systems may beused. In vitro transcription/translation protein expression platforms,that produce sufficient amounts of protein offer many advantages of acell-free protein expression, eliminating the need for laborious up- anddown-stream steps (e.g. host cell transformation, culturing, or lysis)typically associated with cell-based expression systems.

Another aspect of the present invention relates to a method formanufacturing an immunoglobulin or a pharmaceutical preparation thereofcomprising at least one modification in a structural loop region of saidimmunoglobulin and determining the binding of said immunoglobulin to anepitope of an antigen, wherein the unmodified immunoglobulin does notsignificantly bind to said epitope, comprising the steps of:

-   -   providing a nucleic acid encoding an immunoglobulin comprising        at least one loop region,    -   modifying at least one nucleotide residue of at least one of        said loop regions,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with an        epitope,    -   determining whether said modified immunoglobulin binds to said        epitope, and    -   providing the modified immunoglobulin binding to said epitope        and optionally finishing it to a pharmaceutical preparation.

In particular the present invention relates to a method formanufacturing a multi-specific immunoglobulin binding specifically to atleast one first molecule or a pharmaceutical preparation thereofcomprising at least one modification in at least one structural loopregion of said immunoglobulin and determining the specific binding ofsaid at least one loop region to at least one second molecule selectedfrom the group consisting of allergens, tumor associated antigens, selfantigens, enzymes, bacterial antigens, fungal antigens, protozooalantigens and viral antigens, wherein the immunoglobulin containing anunmodified structural loop region does not specifically bind to said atleast one second molecule, comprising the steps of:

-   -   providing a nucleic acid encoding an immunoglobulin binding        specifically to at least one first molecule comprising at least        one structural loop region,    -   modifying at least one nucleotide residue of at least one of        said loop regions encoded by said nucleic acid,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with said at        least one second molecule, and    -   determining whether said modified immunoglobulin binds        specifically to the second molecule and    -   providing the modified immunoglobulin binding specifically to        said at least one second molecule and optionally finishing it to        a pharmaceutical preparation.

The engineering of more than one specificity into a member of a specificbinding pair is preferred (Kufer et al. (2004) Trends in Biotechnologyvol. 22 pages 238-244).

Numerous attempts have been made to produce multi-specific, e.g.bispecific, monoclonal antibodies or antibody fragments. One problem inthe production of bispecific antibodies made of two differentpolypeptide chains (heavy and light chain) is the necessity to expressfour different chains (two heavy and two light chains) in one cellresulting in a number of various combinations of molecules which have tobe separated from the desired bispecific molecule in the mixture. Due totheir similarity the separation of these molecules is difficult andexpensive. A number of techniques have been employed to minimize theoccurrence of such unwanted pairings (Carter (2001) Journal ofImmunological Methods, vol 248, pages 7-15)

One solution to the problem is the production of one polypeptide chainwith two specificities, like e.g. two scFvs linked to each other or theproduction of so-called diabodies. Such molecules have been shown to befar away from the fold of a natural molecule and are notoriouslydifficult to produce (LeGall et al. (2004) Protein Engineering, Design &Selection vol 17 pages 357-366).

Another problem of the current design of bispecific antibodies is thefact that even if the parent antibodies are bi-valently binding to theirrespective binding partner (e.g. IgG), the resulting bispecific antibodyis monovalent for each of the respective binding partner.

The preferred multi-specific molecules of the present invention solvethese problems:

Expression of a bispecific molecule as one polypeptide chain is possible(a modified Ig domain with two binding specificities, see examplesection), which is easier to accomplish than the expression of twoantibody polypeptide chains (Cabilly et al. Proc. Natl. Acad. Sci. USA81:3273-3277 (1984)).

It can also be produced as an antibody like molecule (i.e. made of 2polypeptide chains), due to the fact that the second specificity islocated in the non-variable part of the molecule there is no need fortwo different heavy chains or different light chains. Thus, there is nopossibility of wrong pairing of the two chains.

An antibody of the present invention may consist of a heavy chain and alight chain, which form together a variable region binding to a specificbinding partner the second specificity may be formed by a modified loopof any of the structural loops of either the heavy chain or the lightchain. The binding site may also be formed by more than one non-CDR loopwhich may be structurally neighboured (either on the heavy chain or onthe light chain or on both chains).

The modified antibody or derivative may be a complete antibody or anantibody fragment (e.g. Fab, CH1-CH2, CH2-CH3).

It may bind mono- or multi-valently to binding partners or even withdifferent valency for the different binding partners, depending on thedesign.

As there are a number of various loops available for selection anddesign of a specific binding site in the non-CDR regions of heavy andlight chains it is possible to design antibody derivatives with evenmore than two specificities without the problems mentioned above.

The specific binding domains within one polypeptide chain may beconnected with or without a peptide linker.

Some antibody classes can be regarded as multi-specific, in particularbispecific, by nature: They bind to an antigen (which is typically e.g.either a foreign structure or a cancer associated structure) with thevariable region and bind to Fc-effector molecules with the Fc part (e.g.Fc receptors on various immune cells or complement protein) thusenabling effects such as ADCC, ADCP or CDC.

The Fc-effector molecules are bound by the Fc-part of an immunoglobulinmolecule (for IgG1 it consists of domains CH2 and CH3) and a number ofmethods have been described to optimize effector function by improvementof binding of the Fc-part of an antibody molecule either byglycoengineering techniques (U.S. Pat. No. 6,602,684) or by proteinengineering either directly at the Fc (US 2005/0054832) or indirectly byengineering outside the Fc (US 2005/02444403). Both, binding of the Fcregion to Fc receptor and/or binding to complement proteins such Cq1 hasbeen altered by such techniques. Usually the binding affinity to suchFc-effector molecules is seeked to improve as this correlates withimproved effector functions.

With the current invention it is possible to design antibody binding toFc-effector molecules outside the natural Fc binding region. Modifiedloops in antibody domains other than the loops involved in “natural”Fc-effector molecule binding can be selected from a library or designedto bind to one ore more Fc-effector molecule. An antibody with suchadditional Fc-effector molecule binding sites would either have strongeravidity to a certain Fc-effector molecule or effector-cell displaying anFc-effector molecule and therefore may have an even stronger effect thanglycoengineered antibodies or otherwise improved Fc regions. However,for certain embodiments of the present invention, the effectorcharacteristics of a given antibody to be modified should not directlybe changed but remain unaffected by the modification in the structuralloop according to the present invention.

Antibody fragments have certain advantages as compared to wholeantibodies. Fragments have usually good biodistribution properties andcan more easily be produced. However, most of the antibody fragmentdesigns lack effector functions and have short in vivo half life(Holliger P, et al. Nat Biotechnol. (2005) 23:1126-36.).

Neither CH1 nor Cκ or Cλ domains mediate effector functions which is thereason why Fabs do not show ADCC, ADCP or CDC. The WO 02/44215 describesbinding molecules which consists of the antigen binding site of anantibody and a peptide binding Fc-effector molecules. In such a way anantibody fragment displaying effector functions can be constructed. Thepeptide is being incorporated into the binding molecule at a positionthat does neither destroy the antigen binding nor the ability of thepeptide to bind to an Fc-effector molecule.

According to the present invention however, the binding to Fc-effectormolecules may be performed with modified immunoglobulin domains whichhave been selected for Fc-effector molecule binding from libraries ofrandom loop sequences within a fixed scaffold of an immunoglobulindomain. Therefore, it is possible to select for specific loop sequenceswhich would not bind to Fc-effector molecules outside the Ig-domainscaffold. The polypeptides resulting from the present invention maytherefore preferably consist of more than 100 amino acids.

In order to select for potential effector function of such domainsaccording to the present invention, libraries of mutant CH1, Cκ or Cλdomains may be selected for binding to Fc-receptors and/or complementfactors such as C1q.

In order to increase in vivo half life of a molecule consisting of orcontaining such a domain (e.g. CH1, CH2, CH3, CH4, Cκ or Cλ), binding toFcRn may be selected for with libraries of mutant e.g. CH1-, CH2-, CH3-,CH4-, Cκ- or Cλ-domains according to the present invention.

FcRn-receptors for selection may be provided either on the surface ofcells expressing naturally the respective receptors or by expression andpurification of the extracellular part of the respective receptor. Forthe purpose of this invention a first screening on FcRn may select formutant domains which can further be tested in vitro and even furthercharacterized in FACS experiments by binding to cells expressing FcRnreceptor. It can be further characterized by affinity ranking of bindingto various recombinant FcRn, isoforms and allotypes e.g with surfaceplasmon resonance techniques.

According to a preferred embodiment of the present invention theimmunoglobulin is of human or murine origin.

Since the modified immunoglobulin may be employed for various purposes,in particular in pharmaceutical compositions, the immunoglobulin ispreferably of human or murine origin. Of course, the modifiedimmunoglobulin may also be a humanized or chimeric immunoglobulin.

According to another preferred embodiment of the present invention thehuman immunoglobulin is selected from the group consisting of IgA1,IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM.

The murine immunoglobulin is preferably selected from the groupconsisting of IgA, IgD, IgE, IgG1, IgG2A, IgG2B, IgG2C, IgG3 and IgM.

The modified immunoglobulin may be derived from one of the aboveidentified immunoglobulin classes.

The immunoglobulin comprises preferably a heavy and/or light chain ofthe immunoglobulin or a part thereof.

The modified immunoglobulin may comprise a heavy and/or light chain, atleast one variable and/or constant domain.

The immunoglobulin according to the present invention comprisespreferably at least one constant and/or at least one variable domain ofthe immunoglobulin or a part thereof including a minidomain.

A constant domain is an immunoglobulin fold unit of the constant part ofan immunoglobulin molecule, also referred to as a domain of the constantregion (e.g. CH1, CH2, CH3, CH4, Ck, Cl).

A variable domain is an immunoglobulin fold unit of the variable part ofan immunoglobulin, also referred to as a domain of the variable region(e.g. Vh, Vk, Vl, Vd)

A preferred immunoglobulin according to the invention consists of aconstant domain selected from the group consisting of CH1, CH2, CH3,CH4, Igk-C, Igl-C, or a part thereof including a minidomain, with atleast one loop region, and is characterised in that said at least oneloop region comprises at least one amino acid modification forming atleast one modified loop region, wherein said at least one modified loopregion binds specifically to at least one epitope of an antigen.

Another preferred immunoglobulin according to the invention consists ofa variable domain of a heavy or light chain, or a part thereof includinga minidomain, with at least one loop region, and is characterised inthat said at least one loop region comprises at least one amino acidmodification forming at least one modified loop region, wherein said atleast one modified loop region binds specifically to at least oneepitope of an antigen.

According to a preferred embodiment the constant domain is selected fromthe group of CH1, CH2, CH3, CH4, Igk-C, Igl-C and combinations thereof.

The modified immunoglobulin according to the present invention maycomprise one or more constant domains (e.g. at least two, three, four,five, six, ten domains). If more than one domain is present in themodified immunoglobulin these domains may be of the same type or ofvarying types (e.g. CH1-CH1-CH2, CH3-CH3). Of course also the order ofthe single domains may be of any kind (e.g. CH1-CH3-CH2,CH4-CH1-CH3-CH2).

All numbering of the amino acid sequences of the immunoglobulins isaccording to the IMGT numbering scheme (IMGT, the internationalImMunoGeneTics information; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res. 28: 219-221; Lefranc etal., 2001, Nucleic Acids Res. 29: 207-209; Lefranc et al., 2003, NucleicAcids Res. 31: 307-310; Lefranc et al., 2005, Dev Comp Immunol29:185-203).

According to another preferred embodiment of the present invention themodified loop regions of CH1, CH2, CH3 and CH4 comprise amino acids 7 to21, amino acids 25 to 39, amino acids 41 to 81, amino acids 83 to 85,amino acids 89 to 103 and amino acids 106 to 117.

The loop regions of Igk-C and Igl-C of human origin comprise preferablyamino acids 8 to 18, amino acids 27 to 35, amino acids 42 to 78, aminoacids 83 to 85, amino acids 92 to 100, amino acids 108 to 117 and aminoacids 123 to 126.

The loop regions of Igk-C and Igl-C of murine origin comprise preferablyamino acids 8 to 20, amino acids 26 to 36, amino acids 43 to 79, aminoacids 83 to 85, amino acids 90 to 101, amino acids 108 to 116 and aminoacids 122 to 125.

The structural loop regions of the variable domain of the immunoglobulinof human origin comprise preferably amino acids 8 to 20, amino acids 44to 50, amino acids 67 to 76 and amino acids 89 to 101.

According to a preferred embodiment of the present invention thestructural loop regions of the variable domain of the immunoglobulin ofmurine origin comprise amino acids 6 to 20, amino acids 44 to 52, aminoacids 67 to 76 and amino acids 92 to 101.

The above identified amino acid regions of the respectiveimmunoglobulins comprise loop regions to be modified.

The immunoglobulin according to the invention is preferably of camelorigin.

Camel antibodies comprise only one heavy chain and have the same antigenaffinity as normal antibodies consisting of light and heavy chains.Consequently camel antibodies are much smaller than, e.g., humanantibodies, which allows them to penetrate dense tissues to reach theantigen, where larger proteins cannot. Moreover, the comparativesimplicity, high affinity and specificity and the potential to reach andinteract with active sites, camel's heavy chain antibodies presentadvantages over common antibodies in the design, production andapplication of clinically valuable compounds.

The immunoglobulin of camel origin comprises preferably at least oneconstant domain selected from the group consisting of CH1, CH2 and CH3.

According to a preferred embodiment of the present invention the loopregions of CH1, CH2 and CH3 of the camel immunoglobulin comprise aminoacids 8 to 20, amino acids 24 to 39, amino acids 42 to 78, amino acids82 to 85, amino acids 91 to 103 and amino acids 108 to 117.

According to a preferred embodiment of the present invention thespecific binding of the modified immunoglobulin to the molecule isdetermined by a binding assay selected from the group consisting ofimmunological assays, preferably enzyme linked immunosorbent assays(ELISA), surface plasmon resonance assays, saturation transferdifference nuclear magnetic resonance spectroscopy, transfer NOE (trNOE)nuclear magnetic resonance spectroscopy, competitive assays, tissuebinding assays, live cell binding assays and cellular extract assays.

Binding assays can be carried out using a variety of methods known inthe art, including but not limited to FRET (Fluorescence ResonanceEnergy Transfer) and BRET (Bioluminescence Resonance EnergyTransfer)-based assays, AlphaScreen™ (Amplified Luminescent ProximityHomogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBIACORE®), isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label.

The modified immunoglobulin is preferably conjugated to a label selectedfrom the group consisting of organic molecules, enzyme labels,radioactive labels, colored labels, fluorescent labels, chromogeniclabels, luminescent labels, haptens, digoxigenin, biotin, metalcomplexes, metals, colloidal gold and mixtures thereof.

The modified immunoglobulin may be conjugated to other molecules whichallow the simple detection of said conjugate in, for instance, bindingassays (e.g. ELISA) and binding studies.

Another aspect of the present invention relates to a immunoglobulinconsisting of a constant domain selected from the group consisting ofCH1, CH2, CH3, CH4, Igk-C, Igl-C, or a part thereof includingminidomains, or combinations thereof, with at least one loop region,characterised in that said at least one loop region comprises at leastone amino acid modification forming at least one modified loop region,wherein said at least one modified loop region binds specifically to atleast one epitope of an antigen

It is preferred to combine molecularly at least one modified antibodydomain (=binding to the specific partner via the non-variable sequencesor structural loop) with at least one other binding molecule which canbe an antibody, antibody fragment, a soluble receptor, a ligand oranother modified antibody domain.

The molecule is selected from the group consisting of proteinaceousmolecules, nucleic acids, and carbohydrates.

The loop regions of the modified immunoglobulins may specifically bindto any kind of binding molecules, in particular to proteinaceousmolecules, proteins, peptides, polypeptides, nucleic acids, glycans,carbohydrates, lipids, small organic molecules, anorganic molecules. Ofcourse, the modified immunoglobulins may comprise at least two loopregions whereby each of the loop regions may specifically bind to othermolecules or epitopes.

According to a preferred embodiment of the present invention themolecule binding to the modified structural loop region is selected fromthe group consisting of tumor associated antigens, in particular EpCAM,tumor-associated glycoprotein-72 (TAG-72), tumor-associated antigen CA125, Prostate specific membrane antigen (PSMA), High molecular weightmelanoma-associated antigen (HMW-MAA), tumor-associated antigenexpressing Lewis Y related carbohydrate, Carcinoembryonic antigen (CEA),CEACAM5, HMFG PEM, mucin MUC1, MUC18 and cytokeratin tumor-associatedantigen, bacterial antigens, viral antigens, allergens, fluorescein,lysozyme, toll-like receptor 9, erythropoietin, CD2, CD3, CD3E, CD4,CD11, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30,CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147,GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12,IL-15, IL-18, IL-23, interferon alpha, interferon beta, interferongamma; TNF-alpha, TNFbeta2, TNF.alpha., TNFalphabeta, TNF-R1, TNF-RII,FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT,VEG1, OX40L, TRAIL Receptor-1, A1 Adenosine Receptor, Lymphotoxin BetaReceptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, integrin beta1,integrin beta2, integrin alpha4/beta7, integrin alpha2, integrin alpha3,integrin alpha4, integrin alpha5, integrin alpha6, integrin alphav,alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1, VLA-4,L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor,B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS(B-lymphocyte Stimulator), complement C5, IgE, factor VII, CD64, CBL,NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4),Tissue Factor, VEGF, VEGFR, endothelin receptor, VLA-4, carbohydratessuch as blood group antigens and related carbohydrates,Galili-Glycosylation, Gastrin, Gastrin receptors, tumor associatedcarbohydrates, Hapten NP-cap or NIP-cap, T cell receptor alpha/beta,E-selectin, digoxin, placental alkaline phosphatase (PLAP) andtesticular PLAP-like alkaline phosphatase, transferrin receptor,Heparanase I, human cardiac myosin, Glycoprotein IIb/IIIa (GPIIb/IIIa),human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV,respiratory syncital virus RSV F, RSVF Fgp, VNRintegrin, Hep B gp120,CMV, gpIIbIIIa, HIV IIIB gp120 V3 loop, respiratory syncytial virus(RSV) Fgp, Herpes simplex virus (HSV) gD glycoprotein, HSV gBglycoprotein, HCMV gB envelope glycoprotein, Clostridium perfringenstoxin and fragments thereof.

The modified immunoglobulin according to the present invention maypreferably bind to one of the molecules disclosed above. These moleculescomprise also allergens.

According to another preferred embodiment of the present invention theamino acid residues of positions 15 to 17, 29 to 34, 85.4 to 85.3, 92 to94, 97 to 98 and/or 108 to 110 of CH3 are modified.

The modification of the immunoglobulin according to the presentinvention is preferably a deletion, substitution or an insertion.

According to the present invention at least 1, preferably at least 2, 3,4, 5, 6, 7, 8, 9, 10 and 15, amino acids are deleted, substituted withother amino acids (also with modified amino acids) or inserted into theloop region of the immunoglobulin. However, the maximum number of aminoacids inserted into a loop region of an immunoglobulin may not exceedthe number of 30, preferably 25, more preferably 20, amino acids. Thesubstitution and the insertion of the amino acids occurs preferablyrandomly by methods known in the art and as disclosed in the presentpatent application.

The immunoglobulin according to the invention is according to a specificembodiment characterised in that the CH3 region comprises SEQ ID No. 16or SEQ ID No. 18, when EpCam binds to said immunoglobulin, SEQ ID No.20, when fluorescein binds to said immunoglobulin, SEQ ID No. 22, 24,26, 28, 30 or 32, when lysozyme binds to said immunoglobulin, SEQ ID No.34, 36, 38 or 40, when TLR9 binds to said immunoglobulin, and SEQ ID No.42, when lysozyme and/or erythropoietin bind to said immunoglobulin.

According to a specific embodiment of the invention the immunoglobulinis characterised in that it comprises SEQ ID No. 44 or SEQ ID No. 46,when lysozyme and gp41 bind to said immunoglobulin.

The modified immunoglobulin is preferably conjugated to a label orreporter molecule selected from the group consisting of organicmolecules, enzyme labels, radioactive labels, colored labels,fluorescent labels, chromogenic labels, luminescent labels, haptens,digoxigenin, biotin, metal complexes, metals, colloidal gold andmixtures thereof.

Modified immunoglobulins conjugated to labels as specified above may beused, for instance, in diagnostic methods.

Another aspect of the present invention relates to the use of animmunoglobulin according to the present invention or obtainable by amethod according to the present invention for the preparation of avaccine for active immunization. Hereby the immunoglobulin is eitherused as antigenic drug substance to formulate a vaccine or used forfishing or capturing antigenic structures for use in a vaccineformulation.

Another aspect of the present invention relates to the use of animmunoglobulin according to the present invention or obtainable by amethod according to the present invention for the preparation of aprotein library of immunoglobulins.

Yet another aspect of the present invention relates to a method forspecifically binding and/or detecting a molecule comprising the stepsof:

(a) contacting a modified immunoglobulin according to the presentinvention or a modified immunoglobulin obtainable by a method accordingto the present invention with a test sample suspected to contain saidmolecule, and

(b) detecting the potential formation of a specificimmunoglobulin/molecule complex.

Another aspect of the present invention relates to a method forspecifically isolating a molecule comprising the steps of:

(a) contacting a modified immunoglobulin according to the presentinvention or a modified immunoglobulin obtainable by a method accordingto the present invention with a sample containing said molecule,

(b) separating the specific immunoglobulin/molecule complex formed, and

(c) optionally isolating the molecule from said complex. Theimmunoglobulins according to the present invention may be used toisolate specifically molecules from a sample. If multi-specificimmunoglobulins are used more than one molecules may be isolated from asample. It is especially advantageous using modified immunoglobulins insuch methods because it allows, e.g., to generate a matrix having ahomogeneous surface with defined amounts of binding partners (i.e.Modified immunoglobulins) immobilised thereon which able to bind to themolecules to be isolated. In contrast thereto, if mono-specific bindingpartners are used no homogeneous matrix can be generated because thesingle binding partners do not bind with the same efficiency to thematrix.

Another aspect of the present invention relates to a method fortargeting a compound to a target comprising the steps of:

(a) contacting a modified immunoglobulin according to the presentinvention or a modified immunoglobulin obtainable by a method accordingto the present invention capable to specifically bind to said compound,

(b) delivering the immunoglobulin/compound complex to the target.

Modified immunoglobulins according to the present invention may be usedto deliver at least one compound bound to the CDRs and/or modified loopregions to a target. Such immunoglobulins may be used to targettherapeutic substances to a preferred site of action in the course ofthe treatment of a disease.

Another aspect of the present invention relates to a protein librarycomprising an immunoglobulin according to the present invention orobtainable by the method according to the present invention.

Preferred methods for constructing said library can be found above andin the examples. The library according to the present invention may beused to identify immunoglobulins binding to a distinct molecule.

In particular the present invention relates to the use of a proteinlibrary comprising an immunoglobulin according to the present inventionor obtainable by the method according to the present invention for thedesign of immunoglobulin derivatives. An existing immunoglobulin can bechanged to introduce antigen binding sites into any domain or minidomainby using a protein library of the respective domain of at least 10,preferably 100, more preferably 1000, more preferably 10000, morepreferably 100000, most preferably more than 1000000 variant domainswith at least one modified loop. The library is then screened forbinding to the specific antigen. After molecular characterization forthe desired properties the selected domain or minidomain is cloned intothe original immunoglobulin by genetic engineering techniques so that itreplaces the wild type region. Alternatively, only the DNA coding forthe loops or coding for the mutated amino acids may be exchanged toobtain an immunoglobulin with the additional binding site for thespecific antigen.

The choice of the site for the mutated, antigen-specific structural loopis dependent on the structure of the original immunoglobulin and on thepurpose of the additional binding site. If, for example, the originalmolecule is a complete immunoglobulin which needs to have inserted anadditional antigen binding site without disturbance of the effectorfunction, the loops to be modified would be selected from domainsdistant from CH2 and CH3 which are the natural binding partners toFc-effector molecules. If the original immunoglobulin is a Fab,modification of loops in constant domains of the light chains or theheavy chains or the respective variable domains is possible. To generatea library one may prepare libraries of mutant original molecules whichhave mutations in one ore more structural loops of one or more domains.The selection with complete mutated original molecules may have someadvantages as the selection for antigen binding with a modifiedstructural loop will deliver the sterically advantageous modificationsif tested also for the other properties the mutated immunoglobulinshould show.

The size requirement (i.e. the number of variant proteins) of a proteinlibrary of a mutated domain or a minidomain or a fusion molecule of adomain is dependent on the task. In general, a library to generate anantigen binding site de novo needs to be larger than a library used tofurther modify an already existing engineered antigen binding site madeof a modified structural loop (e.g. for enhancing affinity or changingfine specificity to the antigen).

The present invention also relates to an immunoglobulin library or anucleic acid library comprising a plurality of immunoglobulins, e.g. aconstant or variable domain, a minidomain and/or at least one structuralloop region contained in a minidomain, or nucleic acid moleculesencoding the same. The library contains members with differentmodifications, wherein the plurality is defined by the modifications inthe at least one structural loop region. The nucleic acid librarypreferably includes at least 10 different members (resulting in oneamino acid exchange) and more preferably includes at least 100, morepreferably 1000 or 10000 different members (e.g. designed byrandomisation strategies or combinatory techniques). Even morediversified individual member numbers, such as at least 1000000 or atleast 10000000 are also preferred.

A further aspect of the invention is the combination of two differentdomains or minidomains selected from at least two libraries according tothe invention in order to generate multispecific immunoglobulins. Theseselected specific immunoglobulins may be combined with each other andwith other molecules, similar to building blocks, to design the optimalarrangement of the domains or minidomains to get the desired properties.

Furthermore, one or more modified immunoglobulins according to theinvention may be introduced at various or all the different sites of aprotein possible without destruction of the structure of the protein. Bysuch a “domain shuffling” technique new libraries are created which canagain be selected for the desired properties.

The preferred library contains immunoglobulins according to theinvention, selected from the group consisting of domains of animmunoglobulin, minidomains or derivatives thereof.

A preferred embodiment of the present invention is a binding moleculefor an antigen (antigen binding molecule) comprising at least oneimmunoglobulin domain and a structural loop region being modifiedaccording to the present invention to bind to the antigen, wherein saidbinding molecule does not comprise variable domains of an antibody. Itmay comprise other parts useable for antibody activities (e.g. such asnatural or modified effector regions (sequences); however, it lacks the“natural” binding region of antibodies, i.e. the variable domains intheir naturally occurring position. These antigen binding moleculesaccording to the present invention have the advantages described abovefor the present molecules, yet without the specific binding activity ofantibodies; however with a newly introduced specific binding activity inthe structural loop region.

Preferably, these antigen binding molecules according to the presentinvention comprise CH1, CH2, CH3, CH4, Igk-C, Igl-C and combinationsthereof; said combinations comprising at least two, preferably at leastfour, especially at least six constant domains and at least onestructural loop region modified according to the present invention.Preferably these structural loop regions are either connected viastructural loop region modified according to the present invention orthe structural loops being naturally present between such two constantdomains. An embodiment of these antigen binding molecules according tothe present invention consists of the Fc region of an antibody with atleast one modification in a structural loop according to the presentinvention. Also for the antigen binding molecules according to thepresent invention it is preferred that the new antigen binding sites inthe structural loops are introduced by randomising technologies, i.e. byexchanging one or more amino acid residues of the loop by randomisationtechniques or by introducing randomly generated inserts into suchstructural loops. Alternatively preferred is the use of combinatorialapproaches.

According to another aspect, the present invention relates to a modifiedimmunoglobulin having an antigen binding site foreign to the unmodifiedimmunoglobulin and incorporated in one or more structural loops. Theterm “foreign” means that the antigen binding site is not naturallyformed by the specific region of the immunoglobulin, and a foreignbinding partner, but not the natural binding partner of animmunoglobulin, is bound by the antigen binding site. This means that abinding partner, such as a Fc-receptor or an effector of the immunesystem, is not considered to be bound by the antigen binding siteforeign to the unmodified immunoglobulin.

Preferably, the antigen is selected from the group consisting ofpathogen antigen, tumour associated antigen, enzyme, substrate, selfantigen, organic molecule or allergen. More preferred antigens areselected from the group consisting of viral antigens, bacterial antigensor antigens from pathogens of eukaryote or phages. Preferred viralantigens include HAV-, HBV-, HCV-, HIV I-, HIV II-, Parvovirus-,Influenza-, HSV-, Hepatitis Viruses, Flaviviruses, Westnile Virus, EbolaVirus, Pox-Virus, Smallpox Virus, Measles Virus, Herpes Virus,Adenovirus, Papilloma Virus, Polyoma Virus, Parvovirus, Rhinovirus,Coxsackie virus, Polio Virus, Echovirus, Japanese Encephalitis virus,Dengue Virus, Tick Borne Encephalitis Virus, Yellow Fever Virus,Coronavirus, respiratory syncytial virus, parainfluenza virus, La CrosseVirus, Lassa Virus, Rabies Viruse, Rotavirus antigens; preferredbacterial antigens include Pseudomonas-, Mycobacterium-,Staphylococcus-, Salmonella-, Meningococcal-, Borellia-, Listeria,Neisseria-, Clostridium-, Escherichia-, Legionella-, Bacillus-,Lactobacillus-, Streptococcus-, Enterococcus-, Corynebacterium-,Nocardia-, Rhodococcus-, Moraxella-, Brucella, Campylobacter-,Cardiobacterium-, Francisella-, Helicobacter-, Haemophilus-,Klebsiella-, Shigella-, Yersinia-, Vibrio-, Chlamydia-, Leptospira-,Rickettsia-, Mycobacterium-, Treponema-, Bartonella-antigens. Preferredeukaryotic antigens of pathogenic eukaryotes include antigens fromGiardia, Toxoplasma, Cyclospora, Cryptosporidium, Trichinella, Yeasts,Candida, Aspergillus, Cryptococcus, Blastomyces, Histoplasma,Coccidioides.

Preferred immunoglobulins according to the present invention comprise atleast two antigen binding sites, the first site binding to a firstepitope, and the second site binding to a second epitope.

According to a preferred embodiment, the present immunoglobulincomprises at least two loop regions, the first loop region binding to afirst epitope, and the second loop region binding to a second epitope.Either the at least first or at least second loop region or both may becontain a structural loop. The immunoglobulins according to the presentinventions include the fragments thereof known in the art to befunctional which contain the essential elements according to the presentinvention: the structural loop region modified according to the presentinvention.

Preferably, the immunoglobulin according to the present invention iscomposed of at least two immunoglobulin domains, or a part thereofincluding a minidomain, and each domain contains at least one antigenbinding site.

Also preferred is an immunoglobulin according to the invention, whichcomprises at least one domain of the constant region and/or at least onedomain of the variable region of the immunoglobulin, or a part thereofincluding a minidomain. Thus, a variable domain, which is for examplemodified in the C-terminal region, or the variable domain linked to amodified CH1 region, for instance a modified CH1 minidomain, is one ofthe preferred embodiments.

The preferred immunoglobulin according to the invention comprises adomain that has at least 50% homology with the unmodified domain.

The term “homology” indicates that polypeptides have the same orconserved residues at a corresponding position in their primary,secondary or tertiary structure. The term also extends to two or morenucleotide sequences encoding the homologous polypeptides.

“Homologous immunoglobulin domain” means an immunoglobulin domainaccording to the invention having at least about 50% amino acid sequenceidentity with regard to a full-length native sequence immunoglobulindomain sequence or any other fragment of a full-length immunoglobulindomain sequence as disclosed herein. Preferably, a homologousimmunoglobulin domain will have at least about 50% amino acid sequenceidentity, preferably at least about 55% amino acid sequence identity,more preferably at least about 60% amino acid sequence identity, morepreferably at least about 65% amino acid sequence identity, morepreferably at least about 70% amino acid sequence identity, morepreferably at least about 75% amino acid sequence identity, morepreferably at least about 80% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity to a nativeimmunoglobulin domain sequence, or any other specifically definedfragment of a full-length immunoglobulin domain sequence as disclosedherein.

“Percent (%) amino acid sequence identity” with respect to theimmunoglobulin domain sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific immunoglobulindomain sequence, after aligning the sequence and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.

% amino acid sequence identity values may be obtained as described belowby using the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of theimmunoglobulin domain of interest having a sequence derived from thenative immunoglobulin domain and the comparison amino acid sequence ofinterest (i.e., the sequence against which the immunoglobulin domain ofinterest is being compared which may be the unmodified immunoglobulindomain) as determined by WU-BLAST-2 by (b) the total number of aminoacid residues of the non-randomized parts of the immunoglobulin domainof interest. For example, in the statement “a polypeptide comprising anamino acid sequence A which has or having at least 80% amino acidsequence identity to the amino acid sequence B”, the amino acid sequenceA is the comparison amino acid sequence of interest and the amino acidsequence B is the amino acid sequence of the immunoglobulin domain ofinterest.

In a preferred embodiment the immunoglobulin according to the inventionis a bispecific antibody or a bispecific single chain antibody. Furtherpreferred is that the immunoglobulin comprises a bispecific domain or apart thereof including a minidomain.

The immunoglobulin according to the present invention may be used forany purpose known in the art for immunoglobulins but also enablesapplications which are depending on the combination of specificitiesintroduced by the present invention. Accordingly, the immunoglobulinsaccording to the present inventions are preferably used for therapeuticand prophylactic use (e.g. as an active or passive immunotherapy); forpreparative and analytic use and for diagnostic use.

Another aspect of the present invention relates to a kit of bindingpartners containing

(a) a modified immunoglobulin having an antigen binding site foreign tothe immunoglobulin incorporated in one or more structural loops, and

(b) a binding molecule containing an epitope of said antigen.

Such a binding molecule of this kit according to the present inventionmay be used for identifying the binding specificity of the modifiedimmunoglobulin according to the present invention. By using the bindingmolecule of this kit according to the present invention, the potency ofthe modified immunoglobulins according to the present invention may bedetermined.

Potency as defined here is the binding property of the modified moleculeto its antigen. The binding can be determined quantitatively and/orqualitatively in terms of specificity and/or affinity and/or avidity asused for quality control purposes.

Moreover, the binding molecule of a kit according to the presentinvention may be used for selecting the modified immunoglobulinaccording to the present invention from a library consisting of at least10, preferably at least 100, more preferably at least 1000, morepreferred at least 10000, especially at least 100000 immunoglobulinswith different modifications in the structural loops.

In accordance with the present invention, one of the key features of thepresent invention is that the engineering of the immunoglobulin domainstakes place in regions which are not normally involved in antigenbinding, in other words, in regions other than the CDRs of an antibody.It was observed that the specific fold of immunoglobulin domains allowsthe introduction of random mutations in regions which are structurallyanalogous to the CDRs but different in position in sequence. The regionsidentified by the present invention are, like CDRs, loop regionsconnecting the beta strands of the immunoglobulin fold.

More specifically, it is described herein that by introducing randommutations in the loops connecting beta strands A-B and E-F of a humanIgG1 CH3 domain, mutated CH3 domains were selected that bindspecifically to either Toll like receptor 9-peptide (TLR-9) or to henegg lysozyme, which are a peptide and a protein respectively that arenot normally recognized and bound by human CH3 domains of IgG1. Themutations introduced by us include mutations in which selected aminoacid residues in the wildtype sequence were replaced by randomly chosenresidues, and they also include insertions of extra amino acid residuesin the loops mentioned above.

By analogy the immunoglobulin domains from any class of immunoglobulinsand from immunoglobulins from any species are amenable to this type ofengineering. Furthermore not only the specific loops targeted in thepresent invention can be manipulated, but any loop connecting betastrands in immunoglobulin domains can be manipulated in the same way.

Engineered immunoglobulin domains from any organism and from any classof immunoglobulin can be used according to the present invention eitheras such (as single domains), or as part of a larger molecule. Forexample, they can be part of an intact immunoglobulin, which accordinglywould have its “normal” antigen binding region formed by the 6 CDRs andthe new, engineered antigen binding region. Like this, a multi-specific,e.g. bispecific, immunoglobulin could be generated. The engineeredimmunoglobulin domains can also be part of any fusion protein. The useof these engineered immunoglobulin domains is in the general field ofthe use of immunoglobulins.

The domains of the following immunoglobulins are understood asimmunoglobulin domains here:

for IgG, IgD and IgA: VL, CL, VH, CH1, CH2, CH3

for IgM and IgE: VL, CL, VH, CH1, CH2, CH3, CH4

1. Single immunoglobulin domains randomized on one side, i.e. either inloops connecting beta-strands B-C, D-E or F-G (the “tip”, with theexception of variable domains which are covered by many patents) orbeta-strands A-B, C-D, (C-C′ and C″-D in the case of variable domains)or E-F (the “bottom”). Single loops or any combination of loops can berandomized. Residues can be changed, deleted, or additional residues canbe inserted.

2. Single immunoglobulin domains randomized on both sides, the tip andthe bottom.

3. any protein containing one of the single randomized domains, such as:

-   -   a) “single-chain CH3” dimers (scCH3), scCH2, scCH1/CL,        randomized on one or both sides    -   b) single-chain Fv randomized on the “bottom”, i.e. on the side        opposite to the CDRs    -   c) Fab fragments randomized at the “bottom”, i.e. on the        C-terminal end of the CH1 and of the CL domain    -   d) Fc fragments (i.e. proteins consisting of CH2-CH3) randomized        on one or both sides    -   e) complete immunoglobulins randomized on the bottom of the Fc    -   f) other suitable domains

The primary advantages of the single domains: are very similar to allthe arguments that are used to promote camel VH molecules(“nanobodies”). The randomized immunoglobulin domains are very smallproteins (molecular weight ca. 12-15 kDa, depending on the number ofinserted amino acid residues) and therefore will have the followingadvantages as compared to conventional antibodies or antibody fragmentssuch as scFv and Fabs: recognizing uncommon or hidden epitopes, bindinginto cavities or active sites of protein targets, ease of manufacture,and many others. In the case of an immunoglobulin domain that israndomized on both sides, a bivalent or a bispecific molecule can begenerated. The main advantages of the single domains as part of fusionproteins is additional binding properties can be engineered on any otherprotein.

It is contemplated that any expression system can be used to make theproteins. An analogy to the single domains as described here can befound in the antibodies from the camel, which only has a VH but no VL Inthese proteins, only 3 CDRs (instead of 6 as in “normal” antibodies areresponsible for antigen binding).

The following patent references are incorporated herein by reference asif set forth in their entirety herewith:

U.S. Pat. No. 6,294,654 Modified immunoglobulin molecule incorporatingan antigen in a non-CDR loop region

U.S. Pat. No. 5,844,094 Target binding polypeptide

U.S. Pat. No. 5,395,750 Methods for producing proteins which bind topredetermined antigens

US 2004/0071690 High avidity polyvalent and polyspecific reagents

US 2004/0018508 Surrogate antibodies and methods of preparation and usethereof

US 2003/0157091 Multi-functional proteins

US 2003/0148372 Method to screen phage display libraries with differentligands

US 2002/0103345 Bispecific immunoglobulin-like antigen binding proteinsand method of production

US 2004/0097711 Immunoglobulin superfamily proteins

US 2004/0082508 Secreted proteins

US 2004/0063924 Secreted proteins

US 2004/0043424 Immunoglobulin superfamily proteins

U.S. Pat. No. 5,892,019 Production of a single-gene-encodedimmunoglobulin

U.S. Pat. No. 5,844,094 Target binding polypeptide

DESCRIPTION OF SPECIFIC EXAMPLES Example 1: Construction of the CH3Library and Phage Surface Display

The crystal structure of an IgG1 Fc fragment, which is published in theBrookhaven Database as entry 1OQO.pdb was used to aid in the design ofthe mutated CH3 domain.

The sequence which was used as the basis for construction of the CH3library is given in SEQ ID No. 1. In this sequence, the first amino acidcorresponds to Proline 343 of chain A of Brookhaven database entrylogo.pdb. The last residue contained in logo.pdb is Serine 102 of SEQ IDNo. 1. After detailed analysis of the structure of logo.pdb and byvisual inspection of the residues forming the loops which connect thebeta strands, it was decided to randomize residues 17, 18 and 19, whichare part of the loop connecting beta strand A-B as well as 71, 72, 73,76, and 77, which are part of the loop connecting beta strand E-F of SEQID No. 1. A molecular model of the engineered CH3 domain, with therandomized part indicated by a solvent accessible surface is shown inFIG. 3. The engineered gene was produced by a series of PCR reactionsfollowed by ligation of the resulting PCR products. To facilitateligation, some of the codons of the nucleotide sequence coding for SEQID No. 1 were modified to produce restriction sites without changing theamino acid sequences (silent mutations). For insertion into the cloningvector pHEN1 (Nucleic Acids Res. 1991 Aug. 11; 19(15):4133-7.Multi-subunit proteins on the surface of filamentous phage:methodologies for displaying antibody (Fab) heavy and light chains.Hoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J, Hudson P,Winter G.) in frame with the pelB secretion signal, extra nucleotideresidues encoding Met-Ala were attached at the 5′ end of the sequence tocreate an NcoI restriction site. For the randomized residues, the codonNNS (IUPAC code, where S means C or G) was chosen which encodes all 20naturally occurring amino acids, but avoids 2 out of 3 stop codons. Theengineered sequence is given as a nucleotide sequence in SEQ ID No. 2and as an amino acid sequence in SEQ ID No. 3. The Letter X in SEQ IDNo. 3 denotes randomized amino acid residues. The sequences of the PCRprimers used for assembly of the mutated CH3 domain are given in SEQ IDNo. 4 through 9. FIG. 4 shows a schematic presentation of the PCRfragments generated for assembly of the mutated gene, and the primersused therefor.

cDNA of the heavy chain of the human monoclonal antibody 3D6(Felgenhauer M, Kohl J, Rüker F. Nucleotide sequences of the cDNAsencoding the V-regions of H- and L-chains of a human monoclonal antibodyspecific to HIV-1-gp41. Nucleic Acids Res. 1990 Aug. 25; 18(16):4927)was used as template for the PCR reactions. The 3 PCR products weredigested with SacI and/or HindIII respectively and ligated together. Theligation product was further digested with NcoI and NotI and ligatedinto the surface display phagemid vector pHEN1, which had previouslybeen digested with NcoI and NotI. A number of selected clones werecontrolled by restriction analysis and by DNA sequencing and were foundto contain the insert as planned, including the correctly insertedrandomized sequences. For the following steps of phage preparation,standard protocols were followed. Briefly, the ligation mixture wastransformed into E. coli TG1 cells by electroporation. Subsequently,phage particles were rescued from E. coli TG1 cells with helper phageM13-KO7. Phage particles were then precipitated from culture supernatantwith PEG/NaCl in 2 steps, dissolved in water and used for selection bypanning or, alternatively, they were stored at minus 80° C.

Example 2: Construction of the CH3+3 Library

This library was constructed and cloned in the same way as the CH3library. The amino acid sequence of the construct is given in SEQ ID No.10, the corresponding nucleotide sequence in SEQ ID No. 11, and theprimers used for construction were SEQ ID No. 4-7, SEQ ID No. 9 and SEQID No. 12.

Example 3: Construction of the CH3+5 Library

This library was constructed and cloned in the same way as the CH3library. The amino acid sequence of the construct is given in SEQ ID No.13, the corresponding nucleotide sequence in SEQ ID No. 14, and theprimers used for construction were SEQ ID No. 4-7, SEQ ID No. 9 and SEQID No. 15.

Example 4: Panning of the CH3-Phage Library on TLR-9 Peptide

3 panning rounds were performed according to standard protocols.Briefly, the following method was applied. Maxisorp 96-well plates(Nunc) were coated with a synthetic peptide representing part of thesequence of Toll-like Receptor 9 (TLR-9). 200 μl of the followingsolution were added per well: 0.1M Na-carbonate buffer, pH 9.6, with thefollowing concentrations of dissolved peptide:

1st panning round: 1 mg/ml TLR-9 peptide

2nd panning round: 500 μg/ml TLR-9 peptide

3rd panning round: 100 μg/ml TLR-9 peptide

Incubation was for 1 hour at 37° C., followed by blocking with 2% drymilk (M-PBS) with 200 μl per well for 1 hour at room temperature.

The surface display phage library was then allowed to react with thebound peptide by adding 100 μl phage suspension and 100 μl 4% dry milk(M-PBS), followed by incubation for 45 minutes with shaking and for 90minutes without shaking at room temperature.

Unbound phage particles were washed away as follows. After the 1stpanning round: 10×300 μl T-PBS, 5×300 μl PBS; after the 2nd panninground: 15×300 μl T-PBS, 10×300 μl PBS; after the 3rd panning round:20×300 μl T-PBS, 20×300 PBS.

Elution of bound phage particles was performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension was neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixing 10 ml exponentially growing culture with 0.5 ml elutedphage and incubation for 30 minutes at 37° C. Finally, infected bacteriawere plated on TYE medium with 1% glucose and 100 μg/ml ampicillin, andincubated at 30° C. overnight.

TABLE 1 Results of the panning of the CH3 - phage library on TLR-9peptide (Phage titers) Panning Concentration Input Output round TLR-9 atpanning (phage/ml) (phage/ml) 1st   1 mg/ml 6 × 10¹⁸ 2 × 10¹⁰ 2nd 0.5mg/ml 4 × 10¹⁸ 2 × 10¹⁰ 3rd 0.1 mg/ml 4 × 10²² 6 × 10¹⁰

Example 5: Cloning of Selected Clones of CH3 Mutants Selected AgainstTLR-9 for Soluble Expression

Phagemid DNA from the phage selected through the 3 panning rounds wasisolated with a midi-prep. DNA encoding mutated CH3-regions wasbatch-amplified by PCR and cloned NcoI-NotI into the vectorpNOTBAD/Myc-His, which is the E. coli expression vector pBAD/Myc-His(Invitrogen) with an inserted NotI restriction site to facilitatecloning. Ligated constructs were transformed into E. coli LMG194 cells(Invitrogen) with electroporation, and grown at 30° C. on TYE mediumwith 1% glucose and ampicillin overnight. Selected clones wereinoculated into 200 μl 2×YT medium with ampicillin, grown overnight at30° C., and induced by adding L-arabinose to an end concentration of0.1%. After expression at 16° C. overnight, the cells were harvested bycentrifugation and treated with 100 μl Na-borate buffer, pH 8.0, at 4°C. overnight for preparation of periplasmic extracts. 50 μl of theperiplasmic extracts were used in ELISA (see below).

Example 6: ELISA of CH3 Mutants Selected Against TLR-9 Selected Cloneswere Assayed for Specific Binding to the TLR-9 Peptide by ELISA

Coating: Microtiter plate (NUNC, Maxisorp), 100 μl per well, 20 μg TLR-9peptide/ml 0.1 M Na-carbonate buffer, pH 9.6, 1 h at 37° C.

Wash: 3×200 μl PBS

Blocking: 1% BSA-PBS, 1 h at RT

Wash: 3×200 μl PBS

Periplasmic extract binding: 50 μl periplasmic extract

-   -   50 μl 2% BSA-PBS, at room temperature overnight

Wash: 3×200 μl PBS

1st antibody: anti-His4 (Qiagen), 1:1000 in 1% BSA-PBS, 90 min at RT,100 μl per well

Wash: 3×200 μl PBS

2nd antibody: goat anti mouse*HRP (SIGMA), 1:1000 in 1% BSA-PBS, 90 minat RT, 100 μl per well

Wash: 3×200 μl PBS

Detection: 3 mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5, 0.4 μl30% H₂O₂

Stopping: 100 ml 3M H₂SO₄

Absorbance read: 492/620 nm

Clones that gave a high signal in this first, preliminary ELISA werecultured in a 20-ml volume at the same conditions as described above.Their periplasmic extracts were isolated in 1/20 of the culture volumeas described above and tested with ELISA (as described above) forconfirmation.

TABLE 2 Results of confirmation ELISA with antigen without antigenA_(492/620) A_(492/620) clone 4 readings 1 reading A67 0.0435 0.019 B540.0937 0.051 C67 0.0295 0.013 Background (antigen alone) (12 parallelreadings): 0.0115

Example 7: Panning of the CH3 and of the CH3+5-Phage Library on Hen EggLysozyme

3 panning rounds were performed. Maxisorp 96-well plates (Nunc) werecoated with hen egg lysozyme, by adding 200 μl of the following solutionper well:

PBS, with the following concentrations of dissolved hen egg lysozyme:

1st panning round: 2 mg/ml HEL

2nd panning round: 1 mg/ml HEL

3rd panning round: 1 mg/ml HEL

Incubation was for 1 hour at 37° C., followed by blocking with 2% drymilk (M-PBS) with 200 μl per well for 1 hour at room temperature.

The surface display phage library was then allowed to react with thebound hen egg lysozyme by adding 100 μl phage suspension and 100 μl 4%dry milk (M-PBS), followed by incubation for 45 minutes with shaking andfor 90 minutes without shaking at room temperature.

Unbound phage particles were washed away as follows:

1st panning round: 10×300 μl T-PBS, 5×300 μl PBS

2nd panning round: 15×300 μl T-PBS, 10×300 μl PBS

3rd panning round: 20×300 μl T-PBS, 20×300 μl PBS

Elution of bound phage particles was performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension was neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixture of 10 ml exponentially growing culture with 0.5 mleluted phage and incubation for 30 minutes at 37° C. Finally, infectedbacteria were plated on TYE medium with 1% glucose and 100 μg/mlampicillin, and incubated at 30° C. overnight.

TABLE 3 Results of the panning of phage library CH3 on hen egg lysozyme(Phage titers) Panning Concentration Input Output round HEL at panning(phage/ml) (phage/ml) 1st 2 mg/ml 4.7 × 10¹⁰ 2nd 1 mg/ml 1.29 × 10²² 8.0× 10⁹  3rd 1 mg/ml 5.71 × 10²⁰ 4.8 × 10¹⁰

TABLE 4 Results of the panning of the phage library CH3 + 5 on hen egglysozyme (HEL) (phage titers) Panning Concentration Input Output roundHEL at panning (phage/ml) (phage/ml) 1st 2 mg/ml 8.3 × 10¹⁶ 2.9 × 10⁹2nd 1 mg/ml 2.1 × 10¹⁹ 2.6 × 10⁹ 3rd 1 mg/ml 5.4 × 10¹⁹  1.2 × 10¹⁰

Example 8: Cloning of Selected Clones of Example 7 for SolubleExpression

The cloning of selected clones for soluble expression was performed asdescribed above for the CH3 mutants selected against TLR-9.

Example 9: Soluble Expression of Selected Clones of Example 7

The soluble expression of selected clones was performed as describedabove for the CH3 mutants selected against TLR-9. Periplasmic extractswere tested in a preliminary ELISA (protocol see example 10)

Clones that gave a high signal in this first, preliminary ELISA werecultured in a 20-ml volume at the same conditions as described above.Their periplasmic extracts were isolated in 1/20 of the culture volumeas described above and tested with ELISA (as described in example 10)for confirmation.

Example 10: ELISA of CH3 Mutants Selected Against Hen Egg Lysozyme

Coating: Microtiter plate (NUNC, Maxisorp), 100 μl per well, 100 μg henegg lysozyme/ml in PBS, 1 h at 37° C.

Wash: 3×200 μl PBS

Blocking: 1% BSA-PBS, 1 h at RT

Wash: 3×200 μl PBS

Periplasmic extract binding: 50 μl periplasmic extract

-   -   50 μl 2% BSA-PBS, at room temperature overnight

Wash: 3×200 μl PBS

1st antibody: anti-His4 (Qiagen), 1:1000 in 1% BSA-PBS, 90 min at RT,100 μl per well

Wash: 3×200 μl PBS

2nd antibody: goat anti mouse*HRP (SIGMA), 1:1000 in 1% BSA-PBS, 90 minat RT (room temperature), 100 μl per well

Wash: 3×200 μl PBS

Detection: 3 mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5, 0.4 μl30% H₂O₄

Stopping: 100 ml 3M H₂SO₄

Absorbance read: 492/620 nm

TABLE 5 Results of confirmation ELISA of C_(H)3 mutants selected againsthen egg lysozyme with antigen without antigen A_(492/620) A_(492/620)clone 4 readings 1 reading B12 0.396 0.012 D10 0.415 0.026 D46 0.3980.011 Background (antigen alone) (12 parallel readings): 0.1763

TABLE 6 Results of confirmation ELISA with antigen dilutions of C_(H)3mutants selected against hen egg lysozyme c (μg/ml) clone 200 100 50 2512.5 6.25 3.125 1.55 0.78 0.39 B12 0.707 0.532 0.432 0.297 0.192 0.1500.148 0.049 0.034 0.015 D46 0.713 0.561 0.342 0.220 0.133 0.088 0.0470.032 0.021 0.010 D10 0.715 0.685 0.571 0.368 0.231 0.175 0.171 0.0680.047 0.026 — (nc) 0.449 0.360 0.165 0.072 0.038 0.023 0.017 0.013 0.0090.007 nc: no periplasmic extract added

It is noted that hen egg lysozyme reacts with anti-his₄ antibody,therefore a relatively high background was observed.

TABLE 7 Results of confirmation ELISA of C_(H)3 + 5 mutants selectedagainst hen egg lysozyme with antigen without antigen A_(492/620)A_(492/620) clone 4 readings 1 reading A13 0.197 0.016 A66 0.461 0.019B18 0.533 (5 readings) Not done B20 0.184 0.016 B68 0.535 0.019 B400.706 0.051 C24 0.352 0.072 D22 0.147 0.019 C22 0.439 0.017 D37 0.3600.026 D40 0.559 0.034 D56 0.369 0.019 Background (antigen alone) (12parallel readings): 0.1334 Note: hen egg lysozyme reacts with anti-his₄antibody, therefore a relatively high background was observed.

Example 11: CL Library

Visual inspection of the crystal structure of an Fab fragment (thestructure of the Fab of the human monoclonal antibody 3D6 is used: RSCBProtein Data Bank Entry 1DFB.PDB (He X M, et al. Proc Natl Acad Sci USA.1992 Aug. 1; 89(15):7154-8) and computer-aided analysis (e.g. ProteinExplorer is used for this purpose) of the secondary and tertiarystructure of this protein) allows to identify residues located in loopregions which connect the beta-strands of the CL-domain scaffold. Theseresidues comprise amino acids 8 to 18, amino acids 27 to 35, amino acids42 to 78, amino acids 83 to 85, amino acids 92 to 100, amino acids 108to 117 and amino acids 123 to 126 (numbering according to the IMGTnumbering system (Lefranc M P, et al. Nucleic Acids Res. 2005 Jan. 1; 33(Database issue):D593-7; Lefranc M P, et al. Dev Comp Immunol. 2005;29(3):185-203)).

More specifically, residues 11, 12, 14-18, and 92-95 are randomizedwithin the human CL domain (SEQ ID No. 48). Randomization is achieved byPCR amplification of the coding sequences with PCR primers in which thepositions of the relevant codons are encoded by the nucleotide sequence5′-NNS-3′, which potentially encodes for all 20 amino acids whileavoiding 2 out of 3 stop codons. The library insert is amplified by twoseparate PCR reactions, and the two PCR fragments are ligated togethervia a HpyCH4IV restriction site which is introduced as a silent mutationby the PCR primers. The primers further provide the restrictionendonuclease sites NcoI and NotI respectively for cloning into the phagedisplay vector pHEN (Hoogenboom H R, et al. Nucleic Acids Res. 1991 Aug.11; 19(15):4133-7). The C-terminal cystein of the CL domain is notincluded for the phage display, but can be added later on when amodified CL clone is used e.g. for the construction of an Fab fragment.

As a template for PCR amplification, a plasmid such as pRcCMV-3D6LC(Rüker F, et al. Ann N Y Acad Sci. 1991 Dec. 27; 646:212-9), whichcontains as an insert the complete light chain of the human monoclonalantibody, is used.

For the CL+3 (SEQ ID No. 50, 51) and the CL+5 (SEQ ID No. 52, 53)libraries, which contain additional residues inserted between position92 and 95 of the CL domain, primer CLRHPY3 and CLRHPY5 are usedrespectively instead of primer CLRHPY.

The nucleotide and amino acid sequence of the final product of the PCRsand ligations, cloned into the NcoI site of pHEN1, which leads to theattachment of a pelB leader sequence to the N-terminus of the constructis shown below (SEQ ID No. 48, 49):

 +3  M  K  Y   L  L  P  T   A  A  A   G  L  L   L  L  A  A   1ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC         NcoI  +3  Q  P  A    M  A  V  A  A  P  S   V  F  I   F  P  P  51 CCAGCCGG

CCGTGG CTGCACCATC TGTCTTCATC TTCCCGCCAT  +3S        Q                   A   S  V  V  C   L  L  N 101CTNNSNNSCA GNNSNNSNNS NNSNNSGCCT CTGTTGTGTG CCTGCTGAAT  +3 N  F  Y   P  R  E  A   K  V  Q   W  K  V   D  N  A  L 151 AACTTCTATC CCAGAGAGGC CAAAGTACAG TGGAAGGTGG ATAACGCCCT  +3 Q  S  G    N  S  Q   E  S  V  T  E  Q  D   S  K  D 201 CCAATCGGGT AACTCCCAGG AGAGTGTCAC AGAGCAGGAC AGCAAGGACA                          HpyCH4IV  +3S  T  Y  S   L  S  S   T  L  T   L               Y  E 251GCACCTACAG CCTCAGCAGC ACCCTG

A TGNNSNNSNN SNNSTACGAG  +3 K  H  K   V  Y  A  C   E  V  T   H  Q  G   L  S  S  P 301AAACACAAAG TCTACGCCTG CGAAGTCACC CATCAGGGCC TGAGCTCGCC                NotI  +3   V  T  K   S  F  N   R  G  E  A  A  A 351CGTCACAAAG AGCTTCAACA GGGGAGAG

Primer List for CL Library:

cllnco: (SEQ ID No. 56) 5′-cttaccatgg ccgtggctgc accatctgtc ttcatcttcccgccatctnn snnscagnns nnsnnsnnsn nsgcctctgt tgtgtgc-3′ cllhpy:(SEQ ID No. 57) 5′-tgacaacgtc agggtgctgc tgaggc-3′ clrhpy:(SEQ ID No. 58) 5′-tcagaacgtt gnnsnnsnns nnstacgaga aacacaaagt c-3′clrhpy3: (SEQ ID No. 59) 5′-tcagaacgtt gnnsnnsnns nnsnnsnnsn nstacgagaaacacaaagtc-3′ clrhpy5: (SEQ ID No. 60)5′-tcagaacgtt gnnsnnsnns nnsnnsnnsn nsnnsnnsta cgagaaacac aaagtc-3′clrnot: (SEQ ID No. 61) 5′-catcgoggcc gcctctcccc tgttgaagct c-3′

A number of selected library clones (mutated CL domains cloned in thephagmid vector pHEN1) are controlled by restriction analysis and by DNAsequencing to contain the insert as planned, including the correctlyinserted randomized sequences. For the following steps of phagepreparation, standard protocols are followed. Briefly, the ligationmixture is transformed into E. coli TG1 cells by electroporation.Subsequently, phage particles are rescued from E. coli TG1 cells withhelper phage M13-KO7. Phage particles are then precipitated from culturesupernatant with PEG/NaCl in 2 steps, dissolved in water and used forselection by panning or, alternatively, they can be stored at minus 80°C.

Example 12: CH1 Library

Visual inspection of the crystal structure of an Fab fragment (thestructure of the Fab of the human monoclonal antibody 3D6 is used: RSCBProtein Data Bank Entry 1DFB.PDB) and computer-aided analysis (ProteinExplorer is used for this purpose) of the secondary and tertiarystructure of this protein allows to identify residues located in loopregions which connect the beta-strands of the CH1-domain scaffold. Theseresidues comprise amino acids 7 to 21, amino acids 25 to 39, amino acids41 to 81, amino acids 83 to 85, amino acids 89 to 103 and amino acids106 to 117 (numbering according to the IMGT numbering system).

More specifically, residues 12-19 and 93-100 are randomized within thehuman CH1 domain (SEQ ID No. 54, 55). Randomization is achieved by PCRamplification of the coding sequences with PCR primers in which thepositions of the relevant codons are encoded by the nucleotide sequence5′-NNS-3′, which potentially encodes for all 20 amino acids whileavoiding 2 out of 3 stop codons. The library insert is amplified by twoseparate PCR reactions, and the two PCR fragments are ligated togethervia a BstEII restriction site which occurs naturally in the CH1 domain.The primers further provide the restriction endonuclease sites NcoI andNotI respectively for cloning into the phage display vector pHEN. TheC-terminal cystein of the CH1 domain is not included for the phagedisplay, but can be added later on when a modified CH1 clone is usede.g. for the construction of an Fab fragment.

As a template for PCR amplification, a plasmid such as pRcCMV-3D6HC,which contains as an insert the complete heavy chain of the humanmonoclonal antibody, is used.

The nucleotide and amino acid sequence of the final product of the PCRsand ligations, cloned into the NcoI site of pHEN1, which leads to theattachment of a pelB leader sequence to the N-terminus of the constructis shown below (SEQ ID No. 54, 55):

 +3  M  K  Y   L  L  P  T   A  A  A   G  L  L   L  L  A  A   1ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC          NcoI  +3  Q  P  A   M  A  A   S  T  K  G   P  S  V   F  P  L  51 CCAGCCGG

CCGCCT CCACCAAGGG CCCATCGGTC TTCCCCCTGG  +3 A  P  S  S                            A  L   G  C  L 101CACCCTCCTC CNNSNNSNNS NNSNNSNNSN NSNNSGCCCT GGGCTGCCTG  +3 V  K  D   Y  F  P  E   P  V  T   V  S  W   N  S  G  A 151GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA ACTCAGGCGC  +3  L  T  S   G  V  H   T  F  P  A   V  L  Q   S  S  G 201CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG TCCTCAGGAC                    BstEII  +3 L  Y  S  L   S  S  V   V  T  V   P 251TCTACTCCCT CAGCAGCGT

GTGC CCNNSNNSNN SNNSNNSNNS  +3    T  Y   I  C  N  V   N  H  K   P  S  N   T  K  V  D 301NNSACCTACA TCTGCAACGT GAATCACAAG CCCAGCAACA CCAAGGTGGA                          NotI   +3   K  K  V   E  P  K   S  A  A  A 351CAAGAAAGTT GAGCCCAAAT CT

 A

Primer List for CH1 Library

CH1LNCO: (SEQ ID No. 62) 5′-acgtccatgg ccgcctccac caagggccca tcggtcttccccctggcacc ctcctccnns nnsnnsnnsn nsnnsnnsnn  sgccctgggc tgcctggtc-3′CH1LBST: (SEQ ID No. 63) 5′-ggcacggtca ccacgctgct gag-3′ CH1RBST:(SEQ ID No. 64) 5′-agcgtggtga ccgtgcccnn snnsnnsnns nnsnnsnnsacctacatctg caacgtgaat c-3′ CH1RNOT: (SEQ ID No. 65)5′-catagoggcc gcagatttgg gctcaacttt cttgtc-3′

A number of selected library clones (mutated CH1 domains cloned in thephagmid vector pHEN1) are controlled by restriction analysis and by DNAsequencing to contain the insert as planned, including the correctlyinserted randomized sequences. For the following steps of phagepreparation, standard protocols are followed. Briefly, the ligationmixture is transformed into E. coli TG1 cells by electroporation.Subsequently, phage particles are rescued from E. coli TG1 cells withhelper phage M13-KO7. Phage particles are then precipitated from culturesupernatant with PEG/NaCl in 2 steps, dissolved in water and used forselection by panning or, alternatively, they can be stored at minus 80°C.

Example 13: Panning of the CH1-Phage Library on Hen Egg Lysozyme (HEL)

3 panning rounds are performed with the CH1-phage library (see example12). Maxisorp 96-well plates (Nunc) are coated with hen egg lysozyme, byadding 200 μl of the following solution per well: PBS, with thefollowing concentrations of dissolved hen egg lysozyme:

-   -   1^(st) panning round: 2 mg/ml HEL    -   2^(nd) panning round: 1 mg/ml HEL    -   3^(rd) panning round: 1 mg/ml HEL

Incubation is for 1 hour at 37° C., followed by blocking with 2% drymilk (M-PBS) with 200 μl per well for 1 hour at room temperature.

The surface display phage library is then allowed to react with thebound hen egg lysozyme by adding 100 μl phage suspension and 100 μl 4%dry milk (M-PBS), followed by incubation for 45 minutes with shaking andfor 90 minutes without shaking at room temperature.

Unbound phage particles are washed away as follows:

-   -   1^(st) panning round: 10×300 μl T-PBS, 5×300 μl PBS    -   2^(nd) panning round: 15×300 μl T-PBS, 10×300 μl PBS    -   3^(rd) panning round: 20×300 μl T-PBS, 20×300 μl PBS

Elution of bound phage particles is performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension is neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixture of 10 ml exponentially growing culture with 0.5 mleluted phage and incubation for 30 minutes at 37° C. Finally, infectedbacteria are plated on TYE medium with 1% glucose and 100 μg/mlampicillin, and incubated at 30° C. overnight.

Cloning of Selected Clones of CH1 Mutants Selected Against Lysozyme forSoluble Expression

Phagmid DNA from the phage selected through the 3 panning rounds isisolated with a midi-prep. DNA encoding mutated CH1-domains isbatch-amplified by PCR and cloned NcoI-NotI into the vectorpNOTBAD/Myc-His, which is the E. coli expression vector pBAD/Myc-His(Invitrogen) with an inserted NotI restriction site to facilitatecloning. Ligated constructs are transformed into E. coli LMG194 cells(Invitrogen) with electroporation, and grown at 30° C. on TYE mediumwith 1% glucose and ampicillin overnight. Selected clones are inoculatedinto 200 μl 2×YT medium with ampicillin, grown overnight at 30° C., andinduced by adding L-arabinose to an end concentration of 0.1%. Afterexpression at 16° C. overnight, the cells are harvested bycentrifugation and treated with 100 μl Na-borate buffer, pH 8.0, at 4°C. overnight for preparation of periplasmic extracts. 50 μl of theperiplasmic extracts are used in ELISA.

Clones that give a high signal in this first, preliminary ELISA arecultured in a 20-ml volume at the same conditions as described above.Their periplasmic extracts are isolated in 1/20 of the culture volume asdescribed above and tested with ELISA (as described below) forconfirmation.

ELISA of CH1 Mutants Selected Against Hen Egg Lysozyme

-   -   Coating: Microtiter plate (NUNC, Maxisorp), 100 μl per well, 100        μg hen egg lysozyme/ml in PBS, 1 h at 37° C.    -   Wash: 3×200 μl PBS    -   Blocking: 1% BSA-PBS, 1 h at RT    -   Wash: 3×200 μl PBS    -   Periplasmic extract binding: 50 μl periplasmic extract        -   50 μl 2% BSA-PBS, at room temperature overnight Wash: 3×200            μl PBS    -   1^(st) antibody: anti-His₄ (Qiagen), 1:1000 in 1% BSA-PBS, 90        min at RT, 100 μl per well    -   Wash: 3×200 μl PBS    -   2^(nd) antibody: goat anti mouse*HRP (SIGMA), 1:1000 in 1%        BSA-PBS, 90 min at RT, 100 μl per well    -   Wash: 3×200 μl PBS    -   Detection: 3 mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5,        0.4 μl 30% H₂O₂    -   Stopping: 100 ml 3M H₂SO₄    -   Absorbance read: 492/620 nm

Clones are interpreted as positive when their ELISA signal is at leastthree times that of the background signal.

Example 14: Panning of the CL-Phage Library on Hen Egg Lysozyme (HEL)

3 panning rounds are performed with the CL-phage library (see example11). Maxisorp 96-well plates (Nunc) are coated with hen egg lysozyme, byadding 200 μl of the following solution per well: PBS, with thefollowing concentrations of dissolved hen egg lysozyme:

-   -   1^(st) panning round: 2 mg/ml HEL    -   2^(nd) panning round: 1 mg/ml HEL    -   3^(rd) panning round: 1 mg/ml HEL

Incubation is for 1 hour at 37° C., followed by blocking with 2% drymilk (M-PBS) with 200 μl per well for 1 hour at room temperature.

The surface display phage library is then allowed to react with thebound hen egg lysozyme by adding 100 μl phage suspension and 100 μl 4%dry milk (M-PBS), followed by incubation for 45 minutes with shaking andfor 90 minutes without shaking at room temperature.

Unbound phage particles are washed away as follows:

-   -   1^(st) panning round: 10×300 μl T-PBS, 5×300 μl PBS    -   2^(nd) panning round: 15×300 μl T-PBS, 10×300 μl PBS    -   3^(rd) panning round: 20×300 μl T-PBS, 20×300 μl PBS

Elution of bound phage particles is performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension is neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixture of 10 ml exponentially growing culture with 0.5 mleluted phage and incubation for 30 minutes at 37° C. Finally, infectedbacteria are plated on TYE medium with 1% glucose and 100 μg/mlAmpicillin, and incubated at 30° C. overnight.

Cloning of Selected Clones of CL Mutants Selected Against Lysozyme forSoluble Expression

Phagmid DNA from the phage selected through the 3 panning rounds isisolated with a midi-prep. DNA encoding mutated CL-domains isbatch-amplified by PCR and cloned NcoI-NotI into the vectorpNOTBAD/Myc-His, which is the E. coli expression vector pBAD/Myc-His(Invitrogen) with an inserted NotI restriction site to facilitatecloning. Ligated constructs are transformed into E. coli LMG194 cells(Invitrogen) with electroporation, and grown at 30° C. on TYE mediumwith 1% glucose and ampicillin overnight. Selected clones are inoculatedinto 200 μl 2×YT medium with ampicillin, grown overnight at 30° C., andinduced by adding L-arabinose to an end concentration of 0.1%. Afterexpression at 16° C. overnight, the cells are harvested bycentrifugation and treated with 100 μl Na-borate buffer, pH 8.0, at 4°C. overnight for preparation of periplasmic extracts. 50 μl of theperiplasmic extracts are used in ELISA.

Clones that give a high signal in this first, preliminary ELISA arecultured in a 20-ml volume at the same conditions as described above.Their periplasmic extracts are isolated in 1/20 of the culture volume asdescribed above and tested with ELISA (as described below) forconfirmation.

ELISA of CL Mutants Selected Against Hen Egg Lysozyme

-   -   Coating: Microtiter plate (NUNC, Maxisorp), 100 μl per well, 100        μg hen egg lysozyme/ml in PBS, 1 h at 37° C.    -   Wash: 3×200 μl PBS    -   Blocking: 1% BSA-PBS, 1 h at RT    -   Wash: 3×200 μl PBS    -   Periplasmic extract binding: 50 μl periplasmic extract        -   50 μl 2% BSA-PBS, at room temperature overnight Wash: 3×200            μl PBS    -   1^(st) antibody: anti-His₄ (Qiagen), 1:1000 in 1% BSA-PBS, 90        min at RT, 100 μl per well    -   Wash: 3×200 μl PBS    -   2^(nd) antibody: goat anti mouse*HRP (SIGMA), 1:1000 in 1%        BSA-PBS, 90 min at RT, 100 μl per well    -   Wash: 3×200 μl PBS    -   Detection: 3 mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5,        0.4 μl 30% H₂O₂    -   Stopping: 100 ml 3M H₂SO₄    -   Absorbance read: 492/620 nm

Clones are interpreted as positive when their ELISA signal is at leastthree times that of the background signal.

Example 15: Construction of an Immunoglobulin Domain which is Randomizedon Both Sides (Bispecific Engineered C_(H)3 Domain)

This example describes an engineered immunoglobulin domain with twobinding specificities.

The design of this engineered immunoglobulin domain comprised thefollowing strategy:

-   -   an engineered C_(H)3 domain, clone C24 (see example 10), derived        from the C_(H)3+5 library binding specifically to lysozyme was        used as starting point    -   residues to be randomized were identified in this modified CH3        domain which are connecting β-strands of the immunoglobulin        fold, and which lie on the opposite side of the domain compared        to the residues that were mutated when generating clone C24.    -   PCR primers were designed that allowed randomization of these        residues and synthesis of this engineered immunoglobulin domain        in a procedure similar to the one described above for the        C_(H)3, the C_(H)3+3 and the C_(H)3+5 libraries.

4 PCR products containing randomised positions were ligated andfull-length inserts were amplified by PCR. Subsequently, they werecloned in pHEN-1 via NcoI-NotI sites and transformed into E. coli TG-1cells to construct the library of about 10⁸ colonies. 20 randomly chosencolonies were sequenced and randomised positions were found to beindependently mutated. Also no “wild type” (C24) sequence was observed.The phage library was generated following standard protocols, and aphage titer of 6.32×10¹⁰ TU/ml was achieved.

In order to test bispecificity, recombinant human Erythropoietin (rhEPO)was chosen as second antigen, while it was expected that the constructretained its originally engineered specificity for hen egg lysozyme.rhEPO-reactive phage was selected in 4 panning rounds. In order topreserve the population of C24 clones that after mutagenesis stillshould bind hen egg lysozyme, the first round of selection on rhEPO wasfollowed by a round of panning of the phage population on hen egglysozyme (1 mg/ml in PBS). 200 μl of rhEPO was coated on the 5 wells ofmicrotitre plate (Maxisorp, Nunc) in 0.1 M Na-carbonate buffer, pH 9.6,in decreasing concentrations in subsequent panning rounds (see Tablebelow). After blocking with 2% M-PBS, phage in the blocking agent wasallowed to bind at room temperature for 2 h. After 20 washes with T-PBSand 20 with PBS, it was eluted with 0.1 M glycine, pH 2.2, andneutralised with 2M Tris. Eluted phage was used immediately to infectexponentially growing TG-1. Infected cells were selected onampicilline-containing medium. Phage particles were rescued from culturesupernatants upon superinfection with helper phage M13-KO7, concentratedwith PEG and used in another panning round. Input and output phagenumbers were determined as transforming units of E. coli after everypanning round (Table 8).

TABLE 8 panning phage input phage output round antigen (TU/ml) (TU/ml) 1rhEPO, 500 μg/ml 6.32 × 10¹⁰  1.9 × 10⁵ 2 lysozyme, 1 mg/ml 6.16 × 10¹⁵4.53 × 10¹⁰ 3 rhEPO, 100 μg/ml 6.07 × 10¹⁵ 6.78 × 10¹⁰ 4 rhEPO, 50 μg/ml8.42 × 10¹⁵  3.0 × 10¹¹ 5 rhEPO, 50 μg/ml 5.12 × 10¹⁵ 4.28 × 10¹⁰

Resulting colonies were scraped off the plates, cultured in 2×YT withampicilline and their plasmid DNA was isolated with a midi-prep. Insertswere amplified with a PCR, and then subcloned into vector pNOTBAD andtransformed into an E. coli strain E104. 4×72 colonies were cultured in200 μl 2×YT with ampicilline and induced with 0.1% L-arabinose on thefollowing day. After 24 h expression at 16° C., they were lysed with 200μl Na-borate buffer, pH 8.0 for 6 h at 4° C. and periplasmic extract wasused in ELISA.

For ELISA, Maxisorp plates were coated with hen egg lysozyme in PBS (20μg/ml) or rhEPO in 0.1 M Na-carbonate buffer, pH 9.6, respectively, for1 h at 37° C. After blocking with 1% BSA-PBS, periplasmic extract in thesame blocking agent was allowed to bind overnight. Binding was revealedwith an anti-His-(4) antibody and a goat anti-mouse IgG antibody,conjugated with HRP (for hen egg lysozyme detection) or AP (for rhEPOdetection). Colour reaction of OPD conversion (HRP) was read at 492/620nm after being stopped with 1.25 M H₂SO₄, and pNPP conversion (AP) wasread at 405/620 nm. 14 clones with promising absorbance values wereselected for expression at 20-ml-scale. After 24 h arabinose inductionat 16° C., the cells were collected and lysed overnight in 1 mlNa-borate buffer at 4° C., and the lysate was used for ELISA. ELISA wasperformed as above in 4 parallels, and wells without periplasmic extractand without antigen were used as negative controls. Results (Table 9)were achieved with clone according to SEQ ID No. 42, 43.

TABLE 9 absorbance on no periplasmic antigen binding extract no antigenlysozyme A_(492/620 nm) 0.299 0.110 0.018 rhEPO A_(405/620 nm) 0.2580.095 0.090

Example 16: Engineered C_(H)3 Domains Provide Bispecificity in anFab-Like Format

In the construct used in this example, both the V_(L) and the V_(H)chain of an antibody are fused to an engineered C_(H)3 domain.

The VL and VH region of the human monoclonal antibody 3D6 (He X M, etal. Proc Natl Acad Sci USA. 1992 89:7154-8.; Kohl J, et al. Ann N Y AcadSci. 1991 646:106-14.; Felgenhauer M, et al. Nucleic Acids Res. 199018:4927), which recognizes an epitope on gp41 of HIV-1 was used asfusion partner for the engineered C_(H)3 domain clone C24 which bindsspecifically to hen egg lysozyme.

In order to promote the formation of the VL-CH3/VH-CH3 dimer via adisulfide bond, the residues Ser-Cys were added to the C-terminus of theC24 sequence.

The nucleotide- and amino acid sequences respectively of the two chains,3D6VL-C24 and 3D6VH-C24 are given in SEQ ID No. 47, 46 and SEQ ID No.45, 44, respectively.

Primers were designed that allow the amplification of the codingregions, introducing restriction sites at the same time (silentmutations) which were used to ligate the coding regions together. Forexpression of the genes, the Pichia pastoris expression system waschosen. Constructs were cloned in suitable Pichia pastoris expressionvectors: 3D6VL-C24 was cloned in the pPIC9K (final name: pPIC9K3LC) and3D6VH-C24 (final name: pPICZ3HC) was cloned in pPICZalphaA. ConstructpPICZ3HC was linearized with Bg1 II, transformed into Pichia pastorisGS115 and transformants were selected on zeocin-containing solid medium.One of the transformants was subsequently used as a host cell for theSal I-linearized construct pPIC9K3LC. Double transformants were thenselected on RDB-medium.

Clones were inoculated into 30 ml YPG medium and grown until OD₆₀₀=10,and were then induced by the addition of 1% methanol in BMMY medium. Theinduction was continued for 36 hours at 16° C. Supernatants were removedby centrifugation and were then concentrated about 10-times. Presence ofthe recombinant protein was confirmed by a Western blot with an anti-His(4) antibody, and was estimated to be at a concentration ofapproximately 50-100 μg/l initial culture.

First functional tests were performed with 10×-concentrated supernatant.Firstly, wells of Maxisorp plates were coated with 20 μg/ml hen egglysozyme in PBS or 20 μg/ml epitope of the antibody 3D6 in 0.1 MNa-carbonate buffer, pH 9.6, respectively, for 1 h at 37° C. The 3D6epitope was used in the form of a recombinantly produced GST-fusionprotein. After blocking with 1% BSA-PBS, concentrated supernatants wereallowed to bind overnight in the same blocking agent. Binding wasrevealed with an anti-His (4) antibody and goat anti-mouse antibody,conjugated to HRP, and visualised as colour reaction resulting from OPDconversion at 492/620 nm (Table 10).

TABLE 10 ELISA signal Background Background antigen (A_(492/620)) (noantigen) (no supernatant) lysozyme 0.198 0.003 0.043 3D6 epitope 0.0610.001 0.007

The invention claimed is:
 1. A polypeptide scaffold comprising animmunoglobulin fold of a constant domain comprising six structural loopsconnected by beta strands, three of said six structural loopscorresponding to three structural loops of a human IgG1 constant regionCH3 domain, said three structural loops of said human IgG1 constantregion CH3 domain consisting of the AB loop (positions 17-19 of SEQ IDNO:1), the CD loop (positions 44-47 of SEQ ID NO:1), and the EF loop(positions 71-73 and 76-77 of SEQ ID NO:1), wherein said scaffoldcomprises an amino acid sequence which is 85% identical to amino acids1-104 of SEQ ID NO: 1; and wherein said scaffold comprises a minimumnumber of residues which differ from corresponding residues of SEQ IDNO: 1 selected from the group consisting of: a) wherein two or more ofsaid three structural loops of said scaffold comprise a total of atleast three residues which are different from corresponding residues ofSEQ ID NO: 1; and/or b) wherein one of said three structural loops ofsaid scaffold comprise at least four residues which are different fromcorresponding residues of SEQ ID NO: 1; and wherein said structuralloops of (a) and (b) of said polypeptide scaffold form a solventaccessible surface.
 2. The polypeptide scaffold of claim 1, whereinscaffold comprises an amino acid sequence which is 90% identical toamino acids 1-96 of SEQ ID NO:
 1. 3. The polypeptide scaffold of claim1, wherein scaffold comprises an amino acid sequence which is 95%identical to amino acids 1-96 of SEQ ID NO:
 1. 4. The polypeptidescaffold of claim 1, wherein in (a) each of said two or more of saidthree structural loops of said scaffold comprises at least threeresidues which are different from corresponding residues of SEQ IDNO:
 1. 5. The polypeptide scaffold of claim 1, wherein said structuralloops of (a) and (b) of claim 1 do not comprise an inserted peptide ofpredetermined sequence that specifically binds an epitope independentlyof its insertion into said structural loops of (a) and/or (b).
 6. Thepolypeptide scaffold of claim 1, wherein said scaffold comprises anamino acid sequence selected from the group consisting of: SEQ ID NO:16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42.